Atherosclerosis-associated genes

ABSTRACT

The present invention relates to a combination comprising a plurality of cDNAs which are differentially expressed in cardiovascular diseases. The combination and compositions can be used entirely or in part to diagnose, to stage, to treat, or to monitor the progression or treatment of disorders associated with atherosclerosis.

FIELD OF THE INVENTION

[0001] The invention relates to a combination of isolated cDNAs that aresignificantly co-expressed with one or more knownatherosclerosis-associated genes. The invention also relates to the useof the combination in the diagnosis, prognosis, treatment, andevaluation of therapies for disorders associated with atherosclerosis.

BACKGROUND OF THE INVENTION

[0002] Atherosclerosis is a disorder characterized by cellular changesin the arterial intima and the formation of arterial plaques containingintracellular and extracellular deposits of lipids. The thickening ofartery walls and the narrowing of the arterial lumen underlies thepathologic condition in most cases of coronary artery disease, aorticaneurysm, peripheral vascular disease, and stroke. A number of metabolicpathways and a cascade of molecular events is involved in the cellularmorphogenesis, proliferation, and cellular migration that results inatherogenesis (Libby et al. (1997) Int J Cardiol 62 (S2):23-29).

[0003] The artery walls consist of three layers: the intima (innermost),the media, and the adventitia (outermost). The intima consists of alayer of endothelial cells lining the lumen of arteries and arterioles.Endothelial cells form a barrier against the indiscriminate entry ofsubstances from the blood into the artery. Specific transporter proteinsexpressed by endothelial cells facilitate barrier function. Endothelialcells also secrete a number of substances which help regulate downstreamvascular contractility blood coagulation, and other aspects of vascularbiology. The medial layer of the arterial wall contains smooth musclecells in a matrix of collagen and elastic fibers produced by the smoothmuscle cells. Contraction and relaxation of the smooth muscle layerallows arteries and arterioles to modulate blood pressure and bloodflow. The outermost layer of the arterial wall, the adventitia, is amixture of collagen bundles, elastic fibers, some smooth muscle cells,fibroblasts and nerve cells. The adventitia provides structuralintegrity to the blood vessel and acts as a support matrix for the mediaand intima.

[0004] Initiation of an atherosclerotic lesion often occurs followingvascular endothelial cell injury often attributable to hypertension,diabetes mellitus, hyperlipidemia, fluctuating shear stress, smoking, ortransplant rejection. Nitric oxide and superoxide anions are releasedand react to form cytodestructive peroxynitrite radicals that causeinjury to the endothelium and myocytes of the intima and lead toexpression of a variety of molecules that produce local and systemiceffects. These effects include the release of mediators of inflammationsuch as cytokines, complement components, prostaglandins, and downstreamtranscription factors. Such mediators promote monocyte infiltration ofthe vascular intima and lead to the upregulation of adhesion moleculeswhich encourages attachment of the monocytes to the damaged endothelialcells. Simultaneously, components of the extracellular matrix includingcollagens, fibrinogens, and matrix Gla protein are induced and providesites for monocyte attachment, and annexins, plasminogen activatorinhibitor 1, and nitric oxide synthases are induced to counteract theseeffects.

[0005] Monocytes that infiltrate the lesion accumulate modified lowdensity lipoprotein through scavenger receptors such as CD36 andmacrophage scavenger receptor type I. The abundance of modified lipidsis a factor in atherogenesis and is influenced by modifying enzymes suchas lipoprotein lipase, carboxyl ester lipase, serum amyloid P component,LDL-receptor related protein, microsomal triglyceride transfer protein,and serum esterases such as paraoxonase. Lipid metabolism is governed bycholesterol biosynthesis enzymes such as 3-hydroxy-3-methylglutarylcoenzyme A synthase, and products of the apolipoprotein genes. Modifiedlipid stabilization and accumulation is aided by perilipin andalpha-2-macroglobulin.

[0006] As monocytes accumulate in the lesion, they can rupture andrelease free cholesterol, cytokines, and procoagulants into thesurrounding environment. This is the process that leads to plaquedevelopment; plaque consists of a mass of lipid-engorged monocytes and alipid-rich necrotic core covered by a fibrous cap. The gradualprogression of plaque growth is punctuated by thrombus formation andleads to clinical symptoms such as unstable angina, myocardialinfarction, or stroke. Thrombus formation is initiated by episodicplaque rupture which exposes flowing blood to tissue factors, whichinduce coagulation, and collagen, which activates platelets. Afterinitiation of the atherosclerotic lesion, enzymes that degradeextracellular matrix components (ECM) such as matrix metalloproteinasesand cathepsin K are up-regulated, and inhibitors of ECM aredown-regulated. This results in destabilization of the atheroscleroticlesion and subsequent complications including myocardial infarction,angina, and stroke. Further arterial occlusion and infiltration increasewith the expression of coagulation factors and down-regulation of theirinhibitors, antithrombin III, and lipoprotein-associated coagulationinhibitor.

[0007] Smooth muscle cells build up in the arterial media and constituteone of the principal cell types in atherosclerotic and restenoticlesions. They show a high degree of plasticity and are able to shiftbetween a differentiated, contractile phenotype and a lessdifferentiated, synthetic phenotype. This modulation occurs as aresponse to factors secreted from cells at the site of vascular injuryand results in structural reorganization with a loss of myofilaments andthe formation of an extensive endoplasmic reticulum and a large Golgicomplex. Genes encoding secreted protein, acidic and rich in cysteine(SPARC) and endothelin-1 contribute to these changes. At the same time,the expression of cytoskeletal proteins such as calponin, myosin,desmin, and other gene products in the cells is altered. As a result,the smooth muscle cells lose their contractility and become able tomigrate from the media to the intima, to proliferate, and to secreteextracellular matrix components which contribute to arterial intimalthickening.

[0008] The initiation and progression of atherosclerotic lesiondevelopment requires the interplay of various molecular pathways Manygenes that participate in these processes are known, and some of themhave been shown to have a direct role in atherosclerosis pathogenesis byanimal model experiments, in vitro assays, and epidemiological studies(Krettek et al. (1997) Arterioscler Thromb Vasc Biol 17:2897-2903;Fisher et al. (1997) Atherosclerosis 135:145-159; Shih et al. (1998)Circulation 95:2684-2693; and Bocan et al. (1998) Atherosclerosis139:21-30).

[0009] The present invention satisfies a need in the art by providing acombination comprising a plurality of cDNAs that are useful fordiagnosis, prognosis, treatment, and evaluation of therapies fordisorders associated with atherosclerosis.

SUMMARY OF THE INVENTION

[0010] The invention provides a combination of isolated cDNAs that aresignificantly co-expressed with one or more knownatherosclerosis-associated genes. The combination comprises the isolatedcDNAs having the nucleic acid sequences of SEQ ID NOs:1-25 and thecomplements of SEQ ID NOs:1-25. In one embodiment, the combination isplaced on a substrate. In another embodiment, the substrate is amicroarray.

[0011] The invention also provides a method for using the combination todetect gene expression in a sample containing nucleic acids, the methodcomprising hybridizing the substrate containing the combination to thenucleic acids of the sample under conditions for formation of one ormore hybridization complexes and detecting hybridization complexformation, wherein complex formation indicates gene expression in thesample. In one embodiment, the sample is from artery or is obtainedduring microsurgery to open a blocked artery. In another embodiment,complex formation is compared to standards and is diagnostic of adisorder associated with atherosclerosis.

[0012] The invention further provides a method of using a combination toscreen a plurality of molecules to identify at least one ligand whichspecifically binds a cDNA of the combination, the method comprisingcombining the substrate containing the combination with molecules underconditions to allow specific binding; and detecting specific binding,thereby identifying a ligand which specifically binds at least one cDNAof the combination. In one embodiment, the molecules are selected fromDNA molecules, peptides, proteins, RNA molecules, and transcriptionfactors.

[0013] The invention provides an isolated cDNA comprising apolynucleotide having the nucleic acid sequence of SEQ ID NO:8 and thecomplement of SEQ ID NO:8. In different embodiments, the cDNA is used asa probe, in an expression vector, and in assays for diagnosis,prognosis, and treatment of disorders associated with atherosclerosis.The invention also provides a composition comprising the cDNA and alabeling moiety. The invention further provides a method for using thecDNA to screen a plurality of molecules to identify a ligand whichspecifically binds the cDNA, the method comprising combining the cDNAwith a sample under conditions to allow specific binding; recovering thebound cDNA; and separating the ligand from the bound cDNA, therebyobtaining purified ligand. In one embodiment, the molecules to bescreened are selected from DNA molecules, peptides, proteins, RNAmolecules, and transcription factors. The invention yet further providesa method for using a cDNA to detect gene expression in a samplecontaining nucleic acids, the method comprising hybridizing the cDNA tonucleic acids of a sample under conditions for formation of one or morehybridization complexes; and detecting hybridization complex formation,wherein complex formation indicates gene expression in the sample. Inone embodiment, the cDNA is attached to a substrate. In anotherembodiment, gene expression when compared to standards is diagnostic ofa disorder associated with atherosclerosis.

[0014] The invention provides a vector containing the cDNA, and a hostcell containing the vector. The invention also provides a method forproducing a peptide or protein, the method comprising culturing the hostcell under conditions for expression of the peptide or protein; andrecovering the peptide or protein so produced from cell culture.

[0015] The invention provides a purified peptide or protein comprisingan amino acid sequence expressed by a cDNA of the invention. In oneembodiment, the protein comprises the amino acid sequence of SEQ IDNO:26. The invention additionally provides a composition comprising theprotein and a pharmaceutical carrier. The invention also provides amethod for using a peptide or protein to screen a plurality of moleculesto identify at least one ligand which specifically binds the protein. Inone embodiment, the molecules to be screened are selected from agonists,antagonists, antibodies, DNA molecules, transcription factors, RNAmolecules, and small drug molecules or compounds. The invention furtherprovides a method of using a peptide or protein to purify a ligand.

[0016] The invention provides a method for using the peptide or proteinto produce an antibody which specifically binds the protein. A methodfor preparing polyclonal antibodies comprises immunizing a animal withpeptide or protein under conditions to elicit an antibody response,isolating animal antibodies, attaching the peptide or protein to asubstrate, contacting the substrate with isolated antibodies underconditions to allow specific binding to the peptide or protein,dissociating the antibodies from the peptide or protein, therebyobtaining purified polyclonal antibodies. A method for preparingmonoclonal antibodies comprises immunizing a animal with a peptide orprotein under conditions to elicit an antibody response, isolatingantibody producing cells from the animal, fusing the antibody producingcells with immortalized cells in culture to form monoclonal antibodyproducing hybridoma cells, culturing the hybridoma cells, and isolatingmonoclonal antibodies from culture.

[0017] The invention provides purified antibodies which bindspecifically to a peptide or protein. The invention also provides amethod for using an antibody to detect expression of a peptide orprotein in a sample, the method comprising combining the antibody with asample under conditions for formation of antibody:peptide or proteincomplexes, and detecting complex formation, wherein complex formationindicates expression of the peptide or protein in the sample. In oneaspect, the amount of complex formation when compared to standards isdiagnostic of a disorder of the nervous system.

[0018] The invention provides a method for immunopurification of aprotein comprising attaching an antibody to a substrate, exposing theantibody to a sample containing protein under conditions to allowantibody:protein complexes to form, dissociating the protein from thecomplex, and collecting purified protein. The invention also provides anarray upon which a cDNA encoding a protein, the protein, or an antibodywhich specifically binds the protein are immobilized. The invention alsoprovides a composition comprising a cDNA, a protein, an antibody, or aligand which has agonistic or antagonistic activity.

[0019] The invention provides an antibody comprising an antigen bindingsite, wherein the antigen binding site specifically binds to the proteinThe invention also provides a method for treating a disorder associatedwith the differential expression of a cDNA that is coexpressed with oneor more known atherosclerosis-associated genes in a subject in need, themethod comprising the step of administering to the subject in need theantibody in an amount effective for treating the disorder. The inventionfurther provides an immunoconjugate comprising the antigen binding siteof the antibody or joined to a therapeutic agent. The inventionadditionally provides a method for treating a disorder associated withthe differential expression of a cDNA that is coexpressed with one ormore known atherosclerosis-associated genes in a subject in need, themethod comprising the step of administering to the subject in need theimmunoconjugate in an amount effective for treating the disorder.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING, FIGURE AND TABLES

[0020] The Sequence Listing provides exemplary cDNAs associated withatherosclerosis including polynucleotide sequences SEQ ID NOs:1-25 andthe polypeptide sequence, SEQ ID NO:26. Each sequence is identified by asequence identification number (SEQ ID NO).

[0021]FIG. 1 shows the cDNA having the nucleic acid sequence of SEQ IDNO:8 which encodes the protein having the amino acid sequence of SEQ IDNO:26. The alignment was produced using MAcDNASIS,PRO software (HitachiSoftware Engineering, South San Francisco Calif.).

[0022] Table 4 shows the-co-expression values (-log p) between the knownatherosclerosis-associated genes (abbreviations as shown in Table 3) andthe cDNAs of the invention (SEQ IDs).

[0023] Table 5 summarizes the highly significantly coexpression betweeneach cDNA (SEQ ID) and two known atherosclerosis-associated genes (Gene1 and Gene 2). P-value and the function or importance of the knownatherosclerosis-associated gene are derived from Tables 3 and 4.

[0024] Table 6 shows transcript images for several of the cDNAs (SEQ ID)of the invention. Column 1 shows the SEQ ID; column 2, the library name;column 3, the number of cDNAs in the library; column 4, the descriptionof the sample from which the library was constructed; column 5,transcript abundance; and column 6, percent transcript abundance. Thesesets demonstrate differential expression within experiments usingcardiovascular tissues.

[0025] Table 7 shows microarray data for several of the cDNAs (SEQ ID)of the invention. Column 1 shows the SEQ ID; column 2, the name of themicroarray (GEM) used for the experiment; column 3, the log2 (C5/Cy3ratio); column 4, the description of the Cy3 sample; and column 5, thedescription of the Cy5 sample. These data demonstrate differentialexpression of the cDNAs in experiments using cardiovascular samples.

DESCRIPTION OF THE INVENTION

[0026] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include the plural referenceunless the context clearly dictates otherwise. Thus, for example, areference to “a host cell” includes a plurality of such host cells, anda reference to “an antibody” is a reference to one or more antibodiesand equivalents thereof known to those skilled in the art, and so forth.

[0027] Definitions

[0028] “Antibody” refers to intact immunoglobulin molecule, a polyclonalantibody, a monoclonal antibody, a chimeric antibody, a recombinantantibody, a humanized antibody, single chain antibodies, a Fab fragment,an F(ab′)₂ fragment, an Fv fragment; and an antibody-peptide fusionprotein.

[0029] “Antigenic determinant” refers to an antigenic or immunogenicepitope, structural feature, or region of an oligopeptide, peptide, orprotein which is capable of inducing formation of an antibody whichspecifically binds the protein. Biological activity is not aprerequisite for immunogenicity.

[0030] “Array” refers to an ordered arrangement of at least two cDNAs,proteins, or antibodies on a substrate. At least one of the cDNAs,proteins, or antibodies represents a control or standard, and the otherrepresents a cDNA, protein, or antibody of diagnostic or therapeuticinterest. The arrangement of at least two and up to about 40,000 cDNAs,proteins, or antibodies on the substrate assures that the size andsignal intensity of each labeled complex, formed between each cDNA andat least one nucleic acid, each protein and at least one ligand orantibody, or each antibody and at least one protein to which theantibody specifically binds, is individually distinguishable.

[0031] “Atherosclerosis-associated cDNA” refers to isolatedpolynucleotide that exhibits a statistically significant co-expressionpattern with known atherosclerosis-associated genes. The cDNAs arerepresented by SEQ ID NOs:1-25 of the Sequence Listing and thecomplements of SEQ ID NOs:1-25. They may be of recombinant or syntheticorigin, used in its double-stranded or single-stranded form, andcombined with vitamins, minerals, carbohydrates, lipids, proteins, othernucleic acids, a pharmaceutical carrier or a labeling moiety to performa particular activity or form a useful composition.

[0032] An “atherosclerotic sample or tissue” may be taken using needles,catheters, or scapels and include vessels including the aorta, arteries,arterioles, endothelial cells, plaque, and blood. The sample may containnucleic acids, proteins, antibodies, and the like. Additionally thesample may comprise the soluble fraction of a cell preparation; analiquot of media in which cells were grown; a chromosome, an organelle,or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNAin solution or bound to a substrate.

[0033] A “combination” comprises at least two sequences selected fromSEQ ID NOs:1-25 as presented in the Sequence Listing and the complementsof SEQ ID NOs:1-25.

[0034] “Differential expression” refers to an increased or up-regulatedor a decreased or down-regulated expression as detected by absence,presence, or at least two-fold change in the amount of transcribedmessenger RNA or translated protein in a sample.

[0035] “Disorders associated with atherosclerosis” include anginapectoris, coronary artery disease, myocardial infarction, hypertension,transient cerebral ischemia, mesenteric ischemia, peripheral vasculardisease, renal artery stenosis, and stroke.

[0036] An “expression profile” is a representation of gene expression ina sample. A nucleic acid expression profile is produced usingsequencing, hybridization, or amplification technologies and mRNAs orcDNAs from a sample. A protein expression profile, although timedelayed, mirrors the nucleic acid expression profile and usestwo-dimensional polyacrylamide electrophoresis (2D-PAGE) and massspectrophotometry (MS) or western analysis, enzyme-linked immunosorbentassays (ELISAs), fluorescence activated cell sorting (FACS),radioimmunoassays (RIAs), or arrays and labeling moieties or antibodiesto detect expression in a sample. The nucleic acids, proteins, orantibodies may be used in solution or attached to a substrate, and theirdetection is based on methods and labeling moieties well known in theart.

[0037] A “hybridization complex” is formed between a cDNA of theinvention and a nucleic acid of a sample when the purines of onemolecule hydrogen bond with the pyrimidines of the complementarymolecule, e.g., 5′-A-G-T-C-3′ base pairs with its complete complement,3′-T-C-A-G-5′. The degree of complementarity and the use of nucleotideanalogs affect the efficiency and stringency of hybridization reactions.

[0038] “Identity” as applied to sequences, refers to the quantification(usually percentage) of nucleotide or residue matches between at leasttwo sequences aligned using a standardized algorithm such asSmith-Waterman alignment (Smith and Waterman (1981) J Mol Biol147:195-197), CLUSTALW (Thompson et al. (1994) Nucleic Acids Res22:4673-4680), or BLAST2 (Altschul et al. (1997) Nucleic Acids Res25:3389-340). BLAST2 may be used in a standardized and reproducible wayto insert gaps in one of the sequences in order to optimize alignmentand to achieve a more meaningful comparison between them.

[0039] “Similarity” as applied to proteins uses the same algorithms buttakes into account conservative substitutions of nucleotides orresidues.

[0040] “Isolated or purified” refers to a cDNA or protein that isremoved from its natural environment and that is separated from othercomponents with which it is naturally present.

[0041] “Genes known to be associated with atherosclerosis” include human22 kDa smooth muscle protein, calponin (CNN1), pro alpha 1 (I) collagen(COLIA1), collagen alpha-2 type I (COLIA2), collagen alpha-6 type I(COL6A1), procollagen alpha 2(V) (COL5A2), collagen VI alpha-2 (COL6A2),type VI collagen alpha 3 (COL6A3), pro-alpha-i type 3 collagen (COL3A1),pro-alpha-1(V) collagen (COLSA1), matrix Gla protein (MGP), cathepsin K(CSTK), fibrinogen beta chain gene (FBG), pre-pro-von Willebrand factor(VWF), platelet endothelial cell adhesion molecule (PECAM-1),antithrombin III variant (AT3), lipoprotein lipase (LPL),alpha-2-macroglobulin (A2M), apolipoprotein AI (APOA1), apolipoproteinAII (APOA1)₂, apolipoprotein B-100 (APOB), lipoprotein apoCII (APOC2),pre-apolipoprotein CIII (APOC3), apolipoprotein apo C-IV (APOC4),macrophage scavenger receptor type I (MSR1), human antigen CD36 gene(CD36), serum amyloid P component (SAP), carboxyl ester lipase gene(CEL), paraoxonase 1 (PONI), paraoxonase 2 (PON2), paraoxonase 3 (PON3),perilipin (PLIN), prostaglandin D2 synthase (PTGDS), annexinII/lipocortin II(ANX2), annexin I/lipocortin (ANX1), and secretedprotein, acidic and rich in cysteine (SPARC).

[0042] “Labeling moiety” refers to any reporter molecule whether avisible or radioactive label, stain or dye that can be attached to orincorporated into a cDNA or protein. Visible labels and dyes include butare not limited to anthocyanins, B glucuronidase, BIODIPY, Coomassieblue, Cy3 and Cy5, digoxigenin, FITC, green fluorescent protein,luciferase, spyro red, silver, and the like. Radioactive markers includeradioactive forms of hydrogen, iodine, phosphorous, sulfur, and thelike.

[0043] “Ligand” refers to any agent, molecule, or compound which willbind specifically to a complementary site on a cDNA molecule, apolynucleotide, an epitope or a protein. Such ligands stabilize ormodulate the activity of polynucleotides or proteins and may be composedof inorganic or organic substances including nucleic acids, proteins,carbohydrates, fats, and lipids.

[0044] “Markers for disorders associated with atherosclerosis” refers tocDNAs, peptides or proteins, and antibodies which are useful in thediagnosis, prognosis, treatment, selection or evaluation of therapiesfor disorders associated with atherosclerosis. These markers aredifferentially expressed in samples from subjects predisposed to ormanifesting one of these disorders. The known atherosclerosis-associatedgenes and their contribution and/or function to disorders associatedwith atherosclerosis are listed in TABLE3.

[0045] “Probe” refers to a cDNA of the invention that hybridizes to atleast one nucleic acid in a sample. Where targets are single stranded,probes are complementary single strands. Probes can be labeled for usein hybridization reactions including Southern, northern, in situ, dotblot, array, and like technologies or in screening assays.

[0046] “Protein” refers to a polypeptide or any portion thereof. An“oligopeptide” is an amino acid sequence from about five residues toabout 15 residues that is used as part of a fusion protein to produce anantibody that specifically binds the protein.

[0047] “Specific binding” refers to a special and precise interactionbetween two molecules which is dependent upon their structure,particularly their molecular side groups. For example, the intercalationof a regulatory protein into the major groove of a DNA molecule, thehydrogen bonding along the backbone between two single stranded nucleicacids, or the binding between an epitope of a protein and an agonist,antagonist, or antibody.

[0048] “Substrate” refers to any rigid or semi-rigid support to whichcDNAs or proteins are bound and includes membranes, filters, chips,slides, wafers, fibers, magnetic or nonmagnetic beads, gels, capillariesor other tubing, plates, polymers, and microparticles with a variety ofsurface forms including wells, trenches, pins, channels and pores.

[0049] A “transcript image” (TI) is a profile of gene transcriptionactivity in a particular tissue at a particular time. TI providesassessment of the relative abundance of expressed transcripts in thecDNA libraries of an EST database as described in U.S. Pat. No.5,840,484, incorporated herein by reference.

[0050] “Variant” refers to molecules that are recognized variations of apolynucleotide or a protein. Splice variants may be determined by BLASTscore, wherein the score is at least 100, and most preferably at least400. Allelic variants have a high percent identity to a polynucleotideand may differ by about three bases per hundred bases. “Singlenucleotide polymorphism” (SNP) refers to a change in a single base as aresult of a substitution, insertion or deletion. The change may beconservative (purine for purine) or non-conservative (purine topyrimidine) and may or may not result in a change in an encoded aminoacid.

[0051] The Method

[0052] The present invention encompasses a method for identifying cDNAsthat are significantly co-expressed with knownatherosclerosis-associated genes. In particular, the method identifies acombination of cDNAs useful in diagnosis, prognosis, treatment, andevaluation of therapies for disorders associated with atherosclerosis.

[0053] The method involves identifying cDNAs that are expressed in aplurality of cDNA libraries. These cDNAs include genes of known orunknown function whose expression patterns are compared with geneshaving a known function and disease association to determine whether aspecified coexpression probability threshold is met. Through thiscomparison, a subset of the cDNAs having a high coexpression probabilitywith the known genes can be identified.

[0054] The cDNAs may originate from cDNA libraries derived from avariety of sources including, but not limited to, eukaryotes such ashuman, mouse, rat, dog, monkey, plant, and yeast; prokaryotes such asbacteria; and viruses. The cDNAs can also be selected from a variety ofsequence types including, but not limited to, expressed sequence tags(ESTs), assembled polynucleotide sequences, full length gene codingregions, promoters, introns, enhancers, 5′ untranslated regions, and 3′untranslated regions. To have statistically significant analyticalresults, the cDNAs need to be expressed in at least three cDNAlibraries.

[0055] The cDNA libraries used in the coexpression analysis of thepresent invention can be obtained from adrenal gland, biliary tract,bladder, blood cells, blood vessels, bone marrow, brain, bronchus,cartilage, chromaffin system, colon, connective tissue, cultured cells,embryonic stem cells, endocrine glands, epithelium, esophagus, fetus,ganglia, heart, hypothalamus, immune system, intestine, islets ofLangerhans, kidney, larynx, liver, lung, lymph, muscles, neurons, ovary,pancreas, penis, peripheral nervous system, phagocytes, pituitary,placenta, pleurus, prostate, salivary glands, seminal vesicles,skeleton, spleen, stomach, testis, thymus, tongue, ureter, uterus, andthe like. The number of cDNA libraries selected can range from as few as3 to greater than 10,000. Preferably, the number of the cDNA librariesis greater than 500.

[0056] In a preferred embodiment, the cDNAs are assembled from relatedsequences, such as assembled sequence fragments derived from a singletranscript. Assembly of the sequences can be performed using sequencesof various types including, but not limited to, ESTs, extensions, orshotgun sequences. In a most preferred embodiment, the cDNAs are derivedfrom human sequences that have been assembled using the algorithmdisclosed in U.S. Ser. No. 09/276,534, filed Mar. 25, 1999, incorporatedherein by reference.

[0057] Experimentally, differential expression of the cDNAs can beevaluated by methods including, but not limited to, differential displayby spatial immobilization or by gel electrophoresis, genome mismatchscanning, representational difference analysis, and transcript imaging.Additionally, differential expression can be assessed by microarraytechnology. These methods may be used alone or in combination.

[0058] The known atherosclerosis-associated genes were selected based ontheir function in pathways associated with atherogenesis, their use asdiagnostic or prognostic markers, their behavior in model systems ortheir use as therapeutic targets.

[0059] The procedure for identifying a cDNA that exhibits astatistically significant coexpression pattern with knownatherosclerosis-associated genes is as follows. First, the presence orabsence of a gene in a cDNA library is defined: a gene is present in acDNA library when at least one cDNA fragment corresponding to that geneis detected in a cDNA sample taken from the library, and a gene isabsent from a library when no corresponding cDNA fragment is detected inthe sample.

[0060] Second, the significance of gene coexpression is evaluated usinga probability method to measure a due-to-chance probability of thecoexpression. The probability method can be the Fisher exact test, thechi-squared test, or the kappa test. These tests and examples of theirapplications are well known in the art and can be found in standardstatistics texts (Agresti (1990) Categorical Data Analysis, John Wiley &Sons, New York N.Y.; Rice (1988) Mathematical Statistics and DataAnalysis, Duxbury Press, Pacific Grove Calif.). A Bonferroni correction(Rice, supra, p. 384) can also be applied in combination with one of theprobability methods for correcting statistical results of one genevetsus multiple other genes. In a preferred embodiment, thedue-to-chance probability is measured by a Fisher exact test, and thethreshold of the due-to-chance probability is set preferably to lessthan 0.001, more preferably to less than 0.00001.

[0061] To determine whether two genes, A and B, have similarcoexpression patterns, occurrence data vectors can be generated asillustrated in Table 1. The presence of a gene occurring at least oncein a library is indicated by a one, and its absence from the library, bya zero. TABLE 1 Occurrence data for genes A and B Library 1 Library 2Library 3 . . . Library N gene A 1 1 0 . . . 0 gene B 1 0 1 . . . 0

[0062] For a given pair of genes, the occurrence data in Table 1 can besummarized in a 2×2 contingency table. TABLE 2 Contingency table forco-occurrences of genes A and B Gene A present Gene A absent Total GeneB present 8 2 10 Gene B absent 2 18 20 Total 10 20 30

[0063] Table 2 presents co-occurrence data for gene A and gene B in atotal of 30 libraries. Both gene A and gene B occur 10 times in thelibraries. Table 2 summarizes and presents: 1) the number of times geneA and B are both present in a library; 2) the number of times gene A andB are both absent in a library; 3) the number of times gene A ispresent, and gene B is absent; and 4) the number of times gene B ispresent, and gene A is absent. The upper left entry is the number oftimes the two genes co-occur in a library, and the middle right entry isthe number of times neither gene occurs in a library. The off diagonalentries are the number of times one gene occurs, and the other does not.Both A and B are present eight times and absent 18 times. Gene A ispresent, and gene B is absent, two times; and gene B is present, andgene A is absent, two times. The probability (“p-value”) that the aboveassociation occurs due to chance as calculated using a Fisher exact testis 0.0003. Associations are generally considered significant if ap-value is less than 0.01 (Agresti, supra; Rice, supra).

[0064] This method of estimating the probability for co-expression oftwo genes makes several assumptions. The method assumes that thelibraries are independent and are identically sampled. However, inpractical situations, the selected cDNA libraries are not entirelyindependent, because more than one library may be obtained from a singlesubject or tissue. Nor are they entirely identically sampled, becausedifferent numbers of cDNAs may be sequenced from each library. Thenumber of cDNAs sequenced typically ranges from 5,000 to 10,000 cDNAsper library. In addition, because a Fisher exact co-expressionprobability is calculated for each gene versus 45,233 other assembledgenes, a Bonferroni correction for multiple statistical tests is used.

[0065] The Invention

[0066] The present invention identifies 25 atherosclerosis-associatedcDNAs that exhibit strong association with genes known to bespecifically expressed in atherosclerosis. The results presented inTables 4 and 5 show that the expression of the 25 novelatherosclerosis-associated cDNAs have direct association with theexpression of known atherosclerosis-associated genes as described inTable 3 and in the background of the invention. Therefore, the novelatherosclerosis-associated cDNAs can potentially be used in diagnosis,prognosis, treatment or evaluation of therapies fro disorders associatedwith atherosclerosis. Further, the gene products of the 25 novelatherosclerosis-associated cDNAs are either potential therapeutics ortargets for the development of therapeutics against disorders associatedwith atherosclerosis.

[0067] Therefore, in one embodiment, the present invention encompasses acombination comprising a plurality of cDNAs having the nucleic acidsequences of SEQ ID NOs:1-25 or the complements of SEQ ID NOs:1-25.These 25 cDNAs have been shown by the method of the present invention tohave statistically significant co-expression with knownatherosclerosis-associated genes and with each other. The invention alsoencompasses a cDNA comprising a polynucleotide having the nucleic acidsequence of SEQ ID NO:8 and the complement thereof. As shown in FIG. 1,SEQ ID NO:8 encodes the protein of SEQ ID NO:26. The invention furtherencompasses a protein comprising the polypeptide having the amino acidsequence of SEQ ID NO:26.

[0068] The protein encoded by SEQ ID NO:8 has 366 amino acids. Motifanalyses of SEQ ID NO:26 shows one potential cAMP- and cGMP-dependentprotein kinase phosphorylation site at residue S343, two potentialcasein kinase II phosphorylation sites at residues S179 and T351, andfour potential protein kinase C phosphorylation sites at residues T29,S85, T269, and T324. Additionally, SEQ ID NO:26 contains a potentialsugar transport protein signature sequence from residues L201 to S217.

[0069] cDNAs and Their Uses

[0070] cDNAs can be prepared by a variety of synthetic or enzymaticmethods well known in the art. cDNAs can be synthesized, in whole or inpart, using chemical methods well known in the art (Caruthers et al.(1980) Nucleic Acids Symp Ser (7):215-233). Alternatively, cDNAs can beproduced enzymatically or recombinantly, by in vitro or in vivotranscription.

[0071] Nucleotide analogs can be incorporated into cDNAs by methods wellknown in the art. The only requirement is that the incorporated analogmust base pair with native purines or pyrimidines. For example,2,6-diaminopurine can substitute for adenine and form stronger bondswith thymidine than those between adenine and thymidine. A weaker pairis formed when hypoxanthine is substituted for guanine and base pairswith cytosine. Additionally, cDNAs can include nucleotides that havebeen derivatized chemically or enzymatically.

[0072] cDNAs can be synthesized on a substrate. Synthesis on the surfaceof a substrate may be accomplished using a chemical coupling procedureand a piezoelectric printing apparatus as described by Baldeschweiler etal. (PCT publication WO95/251116). Alternatively, the cDNAs can besynthesized on a substrate surface using a self-addressable electronicdevice that controls when reagents are added as described by Heller etal. (U.S. Pat. No. 5,605,662). cDNAs can be synthesized directly on asubstrate by sequentially dispensing reagents for their synthesis on thesubstrate surface or by dispensing preformed DNA fragments to thesubstrate surface. Typical dispensers include a micropipette deliveringsolution to the substrate with a robotic system to control the positionof the micropipette with respect to the substrate. There can be amultiplicity of dispensers so that reagents can be delivered to thereaction regions efficiently.

[0073] cDNAs can be immobilized on a substrate by covalent means such asby chemical bonding procedures or UV irradiation. In one method, a cDNAis bound to a glass surface which has been modified to contain epoxideor aldehyde groups. In another method, a cDNA is placed on a polylysinecoated surface and UV cross-linked to it as described by Shalon et al.(WO95/35505). In yet another method, a cDNA is actively transported froma solution to a given position on a substrate by electrical means(Heller, supra). cDNAs do not have to be directly bound to thesubstrate, but rather can be bound to the substrate through a linkergroup. The linker groups are typically about 6 to 50 atoms long toprovide exposure of the attached cDNA. Preferred linker groups includeethylene glycol oligomers, diamines, diacids and the like. Reactivegroups on the substrate surface react with a terminal group of thelinker to bind the linker to the substrate. The other terminus of thelinker is then bound to the cDNA. Alternatively, polynucleotides,plasmids or cells can be arranged on a filter. In the latter case, cellsare lysed, proteins and cellular components degraded, and the DNA iscoupled to the filter by UV cross-linking.

[0074] The cDNAs may be used for a variety of purposes. For example, thecombination of the invention may be used on an array. The array, inturn, can be used in high-throughput methods for detecting a relatedpolynucleotide in a sample, screening a plurality of molecules orcompounds to identify a ligand, diagnosing a disorder such as diabetes,or inhibiting or inactivating a therapeutically relevant gene related tothe cDNA.

[0075] When the cDNAs of the invention are employed on an array, thecDNAs are arranged in an ordered fashion so that each cDNA is present ata specified location. Because the cDNAs are at specified locations onthe substrate, the hybridization patterns and intensities, whichtogether create a unique, can be interpreted in terms of expressionlevels of particular genes and can be correlated with a particularmetabolic process, condition, disorder, disease, stage of disease, ortreatment.

[0076] Hybridization

[0077] The cDNAs or fragments or complements thereof may be used invarious hybridization technologies. The cDNAs may be labeled using avariety of reporter molecules by either PCR, recombinant, or enzymatictechniques. For example, a commercially available vector containing thecDNA is transcribed in the presence of an appropriate polymerase, suchas T7 or SP6 polymerase, and at least one labeled nucleotide. Commercialkits are available for labeling and cleanup of such cDNAs. Radioactive(Amersham Biosciences (APB), Piscataway N.J.), fluorescent(Qiagen-Operon, Alameda Calif.), and chemiluminescent labeling (Promega,Madison Wis.) are well known in the art.

[0078] A cDNA may represent the complete coding region of an mRNA or bedesigned or derived from unique regions of the mRNA or genomic molecule,an intron, a 3′ untranslated region, or from a conserved motif. The cDNAis at least 18 contiguous nucleotides in length and is usually singlestranded. Such a cDNA may be used under hybridization conditions thatallow binding only to an identical sequence, a naturally occurringmolecule encoding the same protein, or an allelic variant. Discovery ofrelated human and mammalian sequences may also be accomplished using apool of degenerate cDNAs and appropriate hybridization conditions.Generally, a cDNA for use in Southern or northern hybridizations may befrom about 400 to about 6000 nucleotides long. Such cDNAs have highbinding specificity in solution-based or substrate-based hybridizations.An oligonucleotide, a fragment of the cDNA, may be used to detect apolynucleotide in a sample using PCR.

[0079] The stringency of hybridization is determined by G+C content ofthe cDNA, salt concentration, and temperature. In particular, stringencyis increased by reducing the concentration of salt or raising thehybridization temperature. In solutions used for some membrane basedhybridizations, addition of an organic solvent such as formamide allowsthe reaction to occur at a lower temperature. Hybridization may beperformed with buffers, such as 5×saline sodium citrate (SSC) with 1%sodium dodecyl sulfate (SDS) at 60° C., that permit the formation of ahybridization complex between nucleic acid sequences that contain somemismatches. Subsequent washes are performed with buffers such as 0.2×SSCwith 0.1% SDS at either 45° C. (medium stringency) or 65°-68° C. (highstringency). At high stringency, hybridization complexes will remainstable only where the nucleic acids are completely complementary. Insome membrane-based hybridizations, preferably 35% or most preferably50%, formamide may be added to the hybridization solution to reduce thetemperature at which hybridization is performed. Background signals maybe reduced by the use of detergents such as Sarkosyl or TRITON X-100(Sigma-Aldrich, St. Louis Mo.) and a blocking agent such as denaturedsalmon sperm DNA. Selection of components and conditions forhybridization are well known to those skilled in the art and arereviewed in Ausubel et al. (1997, Short Protocols in Molecular Biology,John Wiley & Sons, New York N.Y., Units 2.8-2.11, 3.18-3.19 and4.6-4.9).

[0080] Dot-blot, slot-blot, low density and high density arrays areprepared and analyzed using methods known in the art. cDNAs from about18 consecutive nucleotides to about 5000 consecutive nucleotides inlength are contemplated by the invention and used in array technologies.The preferred number of cDNAs on an array is at least about 100,000, amore preferred number is at least about 40,000, an even more preferrednumber is at least about 10,000, and a most preferred number is at leastabout 600 to about 800. The array may be used to monitor the expressionlevel of large numbers of genes simultaneously and to identify geneticvariants, mutations, and SNPs. Such information may be used to determinegene function; to understand the genetic basis of a disorder; todiagnose a disorder; and to develop and monitor the activities oftherapeutic agents being used to control or cure a disorder. (See, e.g.,U.S. Pat. No. 5,474,796; WO95/11995; WO95/35505; U.S. Pat. No.5,605,662; and U.S. Pat. No. 5,958,342.)

[0081] Screening and Purification Assays Using cDNAs

[0082] A cDNA may be used to screen a library or a plurality ofmolecules or compounds for a ligand which specifically binds the cDNA.Ligands may be DNA molecules, RNA molecules, peptide nucleic acidmolecules, peptides, proteins such as transcription factors, promoters,enhancers, repressors, and other proteins that regulate replication,transcription, or translation of the polynucleotide in the biologicalsystem. The assay involves combining the cDNA or a fragment thereof withthe molecules or compounds under conditions that allow specific bindingand detecting the bound cDNA to identify at least one ligand thatspecifically binds the cDNA.

[0083] In one embodiment, the cDNA may be incubated with a library ofisolated and purified molecules or compounds and binding activitydetermined by methods such as a gel-retardation assay (U.S. Pat. No.6,010,849) or a reticulocyte lysate transcriptional assay. In anotherembodiment, the cDNA may be incubated with nuclear extracts frombiopsied and/or cultured cells and tissues. Specific binding between thecDNA and a molecule or compound in the nuclear extract is initiallydetermined by gel shift assay. Protein binding may be confirmed byraising antibodies against the protein and adding the antibodies to thegel-retardation assay where specific binding will cause a supershift inthe assay.

[0084] In another embodiment, the cDNA may be used to purify a moleculeor compound using affinity chromatography methods well known in the art.In one embodiment, the cDNA is chemically reacted with cyanogen bromidegroups on a polymeric resin or gel. Then a sample is passed over andreacts with or binds to the cDNA. The molecule or compound which isbound to the cDNA may be released from the cDNA by increasing the saltconcentration of the flow-through medium and collected.

[0085] The cDNA may be used to purify a ligand from a sample. A methodfor using a cDNA to purify a ligand would involve combining the cDNA ora fragment thereof with a sample under conditions to allow specificbinding, recovering the bound cDNA, and using an appropriate agent toseparate the cDNA from the purified ligand.

[0086] Protein Production and Uses

[0087] The full length cDNAs or fragments thereof may be used to producepurified proteins using recombinant DNA technologies described hereinand taught in Ausubel (supra; Units 16.1-16.62). One of the advantagesof producing proteins by these procedures is the ability to obtainhighly-enriched sources of the proteins thereby simplifying purificationprocedures.

[0088] The proteins may contain amino acid substitutions, deletions orinsertions made on the basis of similarity in polarity, charge,solubility, hydrophobicity, hydrophilicity, and/or the amphipathicnature of the residues involved. Such substitutions may be conservativein nature when the substituted residue has structural or chemicalproperties similar to the original residue (e.g., replacement of leucinewith isoleucine or valine) or they may be nonconservative when thereplacement residue is radically different (e.g., a glycine replaced bya tryptophan). Computer programs included in LASERGENE software(DNASTAR, Madison Wis.) and algorithms included in RasMol software(University of Massachusetts, Amherst Mass.) may be used to helpdetermine which and how many amino acid residues in a particular portionof the protein may be substituted, inserted, or deleted withoutabolishing biological or immunological activity.

[0089] Expression of Encoded Proteins

[0090] Expression of a particular cDNA may be accomplished by cloningthe cDNA into a vector and transforming this vector into a host cell.The cloning vector used for the construction of cDNA libraries in theLIFESEQ databases (Incyte Genomics, Palo Alto Calif.) may also be usedfor expression. Such vectors usually contain a promoter and a polylinkeruseful for cloning, priming, and transcription. An exemplary vector mayalso contain the promoter for β-galactosidase, an amino-terminalmethionine and the subsequent seven amino acid residues ofβ-galactosidase. The vector may be transformed into competent E. colicells. Induction of the isolated bacterial strain withisopropylthiogalactoside (IPTG) using standard methods will produce afusion protein that contains an N terminal methionine, the first sevenresidues of β-galactosidase, about 15 residues of linker, and theprotein encoded by the cDNA.

[0091] The cDNA may be shuttled into other vectors known to be usefulfor expression of protein in specific hosts. Oligonucleotides containingcloning sites and fragments of DNA sufficient to hybridize to stretchesat both ends of the cDNA may be chemically synthesized by standardmethods. These primers may then be used to amplify the desired fragmentsby PCR. The fragments may be digested with appropriate restrictionenzymes under standard conditions and isolated using gelelectrophoresis. Alternatively, similar fragments are produced bydigestion of the cDNA with appropriate restriction enzymes and filled inwith chemically synthesized oligonucleotides. Fragments of the codingsequence from more than one gene may be ligated together and expressed.

[0092] Signal sequences that dictate secretion of soluble proteins areparticularly desirable as component parts of a recombinant sequence. Forexample, a chimeric protein may be expressed that includes one or moreadditional purification-facilitating domains. Such domains include, butare not limited to, metal-chelating domains that allow purification onimmobilized metals, protein A domains that allow purification onimmobilized immunoglobulin, and the domain utilized in the FLAGSextension/affinity purification system (Immunex, Seattle Wash.). Theinclusion of a cleavable-linker sequence such as ENTEROKINASEMAX(Invitrogen, San Diego Calif.) between the protein and the purificationdomain may also be used to recover the protein.

[0093] Suitable host cells may include, but are not limited to,mammalian cells such as Chinese Hamster Ovary (CHO) and human 293 cells,insect cells such as Sf9 cells, plant cells such as Nicotiana tabacum,yeast cells such as Saccharomyces cerevisiae, and bacteria such as E.coli. For each of these cell systems, a useful vector may also includean origin of replication and one or two selectable markers to allowselection in bacteria as well as in a transformed eukaryotic host.Vectors for use in eukaryotic host cells may require the addition of 3′poly(A) tail if the cDNA lacks poly(A).

[0094] Additionally, the vector may contain promoters or enhancers thatincrease gene expression. Many promoters are known and used in the art.Most promoters are host specific and exemplary promoters includes SV40promoters for CHO cells; T7 promoters for bacterial hosts; viralpromoters and enhancers for plant cells; and PGH promoters for yeast.Adenoviral vectors with the rous sarcoma virus enhancer or retroviralvectors with long terminal repeat promoters may be used to drive proteinexpression in mammalian cell lines. Once homogeneous cultures ofrecombinant cells are obtained, large quantities of secreted solubleprotein may be recovered from the conditioned medium and analyzed usingchromatographic methods well known in the art. An alternative method forthe production of large amounts of secreted protein involves thetransformation of mammalian embryos and the recovery of the recombinantprotein from milk produced by transgenic cows, goats, sheep, and thelike.

[0095] In addition to recombinant production, proteins or portionsthereof may be produced manually, using solid-phase techniques (Stewartet al. (1969) Solid-Phase Peptide Synthesis, W H Freeman, San FranciscoCalif.; Merrifield (1963) J Am Chem Soc 5:2149-2154), or using machinessuch as the 431A peptide synthesizer (Applied Biosystems (ABI), FosterCity Calif.). Proteins produced by any of the above methods may be usedas pharmaceutical compositions to treat disorders associated with nullor inadequate expression of the genomic sequence.

[0096] Screening and Purification Assays Using Proteins

[0097] A protein or a portion thereof encoded by the cDNA may be used toscreen a library or a plurality of molecules or compounds for a ligandwith specific binding affinity or to purify a molecule or compound froma sample. The protein or portion thereof employed in such screening maybe free in solution, affixed to an abiotic or biotic substrate, orlocated intracellularly. For example, viable or fixed prokaryotic hostcells that are stably transformed with recombinant nucleic acids thathave expressed and positioned a protein on their cell surface can beused in screening assays. The cells are screened against a library or aplurality of ligands and the specificity of binding or formation ofcomplexes between the expressed protein and the ligand may be measured.The ligands may be agonists, antagonists, antibodies, DNA molecules,enhancers, small drug molecules, immunoglobulins, inhibitors, mimetics,peptide nucleic acid molecules, peptides, pharmaceutical agents,proteins, and regulatory proteins, repressors, RNA molecules, ribozymes,transcription factors, or any other test molecule or compound thatspecifically binds the protein. An exemplary assay involves combiningthe mammalian protein or a portion thereof with the molecules orcompounds under conditions that allow specific binding and detecting thebound protein to identify at least one ligand that specifically bindsthe protein.

[0098] This invention also contemplates the use of competitive drugscreening assays in which neutralizing antibodies capable of binding theprotein specifically compete with a test compound capable of binding tothe protein or oligopeptide or fragment thereof. One method for highthroughput screening using very small assay volumes and very smallamounts of test compound is described in U.S. Pat. No. 5,876,946.Molecules or compounds identified by screening may be used in a modelsystem to evaluate their toxicity, diagnostic, or therapeutic potential.

[0099] The protein may be used to purify a ligand from a sample. Amethod for using a protein to purify a ligand would involve combiningthe protein or a portion thereof with a sample under conditions to allowspecific binding, recovering the bound protein, and using an appropriatechaotropic agent to separate the protein from the purified ligand.

[0100] Production of Antibodies

[0101] A protein encoded by a cDNA of the invention may be used toproduce specific antibodies. Antibodies may be produced using anoligopeptide or a portion of the protein with inherent immunologicalactivity. Methods for producing antibodies include: 1) injecting ananimal, usually goats, rabbits, or mice, with the protein, or anantigenically-effective portion or an oligopeptide thereof, to induce animmune response; 2) engineering hybridomas to produce monoclonalantibodies; 3) inducing in vivo production in the lymphocyte population;or 4) screening libraries of recombinant immunoglobulins. Recombinantimmunoglobulins may be produced as taught in U.S. Pat. No. 4,816,567.

[0102] Antibodies produced using the proteins of the invention areuseful for the diagnosis of prepathologic disorders as well as thediagnosis of chronic or acute diseases characterized by abnormalities inthe expression, amount, or distribution of the protein. A variety ofprotocols for competitive binding or immunoradiometric assays usingeither polyclonal or monoclonal antibodies specific for proteins arewell known in the art. Immunoassays typically involve the formation ofcomplexes between a protein and its specific binding molecule orcompound and the measurement of complex formation. Immunoassays mayemploy a two-site, monoclonal-based assay that utilizes monoclonalantibodies reactive to two noninterfering epitopes on a specific proteinor a competitive binding assay (Pound (1998) Immunochemical Protocols,Humana Press, Totowa N.J.).

[0103] Immunoassay procedures may be used to quantify expression of theprotein in cell cultures, in subjects with a particular disorder or inmodel animal systems under various conditions. Increased or decreasedproduction of proteins as monitored by immunoassay may contribute toknowledge of the cellular activities associated with developmentalpathways, engineered conditions or diseases, or treatment efficacy. Thequantity of a given protein in a given tissue may be determined byperforming immunoassays on freeze-thawed detergent extracts ofbiological samples and comparing the slope of the binding curves tobinding curves generated by purified protein.

[0104] Antibody Arrays

[0105] In an alternative to yeast two hybrid system analysis ofproteins, an antibody array can be used to study protein-proteininteractions and phosphorylation. A variety of protein ligands areimmobilized on a membrane using methods well known in the art. The arrayis incubated in the presence of cell lysate until protein:antibodycomplexes are formed. Proteins of interest are identified by exposingthe membrane to an antibody specific to the protein of interest. In thealternative, a protein of interest is labeled with digoxigenin (DIG) andexposed to the membrane; then the membrane is exposed to anti-DIGantibody which reveals where the protein of interest forms a complex.The identity of the proteins with which the protein of interestinteracts is determined by the position of the protein of interest onthe membrane.

[0106] Antibody arrays can also be used for high-throughput screening ofrecombinant antibodies. Bacteria containing antibody genes arerobotically-picked and gridded at high density (up to 18,342 differentdouble-spotted clones) on a filter. Up to 15 antigens at a time are usedto screen for clones to identify those that express binding antibodyfragments. These antibody arrays can also be used to identify proteinswhich are differentially expressed in samples (de Wildt et al. (2000)Nature Biotechnol 18:989-94).

[0107] Labeling of Molecules for Assay

[0108] A wide variety of reporter molecules and conjugation techniquesare known by those skilled in the art and may be used in various cDNA,polynucleotide, protein, peptide or antibody assays. Synthesis oflabeled molecules may be achieved using commercial kits forincorporation of a labeled nucleotide such as ³²P-dCTP, Cy3-dCTP orCy5-dCTP or amino acid such as ³⁵S-methionine. Polynucleotides, cDNAs,proteins, or antibodies may be directly labeled with a reporter moleculeby chemical conjugation to amines, thiols and other groups present inthe molecules using reagents such as BIODIPY or FITC (Molecular Probes,Eugene Oreg.).

[0109] The proteins and antibodies may be labeled for purposes of assayby joining them, either covalently or noncovalently, with a reportermolecule that provides for a detectable signal. A wide variety of labelsand conjugation techniques are known and have been reported in thescientific and patent literature including, but not limited to U.S. Pat.No. 3,817,837; U.S. Pat. No. 3,850,752; U.S. Pat. No. 3,939,350; U.S.Pat. No. 3,996,345; U.S. Pat. No. 4,277,437; U.S. Pat. No. 4,275,149;and U.S. Pat. No. 4,366,241.

[0110] Diagnostics

[0111] The cDNAs, or fragments thereof, may be used to detect andquantify differential gene expression; absence, presence, or excessexpression of mRNAs; or to monitor mRNA levels during therapeuticintervention. Disorders associated with atherosclerosis include anginapectoris, coronary artery disease, myocardial infarction, hypertension,transient cerebral ischemia, mesenteric ischemia, peripheral vasculardisease, renal artery stenosis, and stroke. These cDNAs can also beutilized as markers of treatment efficacy against the disorders notedabove and other disorders, conditions, and diseases over a periodranging from several days to months. The diagnostic assay may usehybridization or amplification technology to compare gene expression ina biological sample from a patient to standard samples in order todetect altered gene expression. Qualitative or quantitative methods forthis comparison are well known in the art.

[0112] For example, the cDNA may be labeled by standard methods andadded to a biological sample from a patient under conditions forhybridization complex formation. After an incubation period, the sampleis washed and the amount of label (or signal) associated withhybridization complexes is quantified and compared with a standardvalue. If the amount of label in the patient sample is significantlyaltered in comparison to the standard value, then the presence of theassociated condition, disease or disorder is indicated.

[0113] In order to provide a basis for the diagnosis of a condition,disease or disorder associated with gene expression, a normal orstandard expression profile is established. This may be accomplished bycombining a biological sample taken from normal subjects, either animalor human, with a probe under conditions for hybridization oramplification. Standard hybridization may be quantified by comparing thevalues obtained using normal subjects with values from an experiment inwhich a known amount of a purified target sequence is used. Standardvalues obtained in this manner may be compared with values obtained fromsamples from patients who are symptomatic for a particular condition,disease, or disorder. Deviation from standard values toward thoseassociated with a particular condition is used to diagnose thatcondition.

[0114] Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies and inclinical trial or to monitor the treatment of an individual patient.Once the presence of a condition is established and a treatment protocolis initiated, diagnostic assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in a normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0115] Gene Expression Profiles

[0116] A gene expression profile comprises a plurality of cDNAs and aplurality of detectable hybridization complexes, wherein each complex isformed by hybridization of one or more probes to one or morecomplementary nucleic acids in a sample. The cDNAs of the invention areused as elements on a array to analyze gene expression profiles. In oneembodiment, the array is used to monitor the progression of disease.Researchers or clinicians can catalog the differences in gene expressionbetween healthy and diseased tissues or cells. By analyzing changes inpatterns of gene expression, disease can be diagnosed at earlier stagesbefore the patient is symptomatic. The invention can be used toformulate a prognosis and to design a treatment regimen. The inventioncan also be used to monitor the efficacy of treatment. For treatmentswith known side effects, the array is employed to improve the treatmentregimen. A dosage is established that causes a change in geneticexpression patterns indicative of successful treatment. Expressionpatterns associated with the onset of undesirable side effects areavoided. This approach may be more sensitive and rapid than waiting forthe patient to show inadequate improvement, or to manifest side effects,before altering the course of treatment.

[0117] Experimentally, expression profiles can also be evaluated bymethods including, but not limited to, differential display by spatialimmobilization or by gel electrophoresis, labeling with radionuclide andquantification using a scintillation counter, genome mismatch scanning,representational difference analysis, transcript imaging, quantitativePCR, and by protein or antibody arrays. Expression profiles produced bythese methods may be contrasted with expression profiles produced usingnormal or diseased tissues. Of note is the correspondence between mRNAand protein expression has been discussed by Zweiger (2001, Transducingthe Genome. McGraw-Hill, San Francisco, Calif.) and Glavas et al. (2001;T cell activation upregulates cyclic nucleotide phosphodiesterases 8A1and 7A3, Proc Natl Acad Sci 98:6319-6342) among others.

[0118] In another embodiment, animal models which mimic a human diseasecan be used to produce expression profiles associated with a particularcondition, disorder or disease; or treatment of the condition, disorderor disease. Novel treatment regimens may be tested in these animalmodels using arrays to establish and then follow expression profilesover time. In addition, arrays may be used with cell cultures or tissuesremoved from animal models to rapidly screen large numbers of candidatedrug molecules, looking for ones that produce an expression profilesimilar to those of known therapeutic drugs, with the expectation thatmolecules with the same expression profile will likely have similartherapeutic effects. Thus, the invention provides the means to rapidlydetermine the molecular mode of action of a drug.

[0119] Assays Using Antibodies

[0120] Antibodies directed against antigenic determinants of a proteinencoded by a cDNA of the invention may be used in assays to quantify theamount of protein found in a particular human cell. Such assays includemethods utilizing the antibody and a label to detect expression levelunder normal or disease conditions. The antibodies may be used with orwithout modification, and labeled by joining them, either covalently ornoncovalently, with a labeling moiety.

[0121] Protocols for detecting and measuring protein expression usingeither polyclonal or monoclonal antibodies are well known in the art.Examples include, but are not limited to, western analysis, ELISA, RIA,FACS, and arrays. Such immunoassays typically involve the formation ofcomplexes between the protein and its specific antibody and themeasurement of such complexes. These assays are specifically describedin Pound (supra).

[0122] Therapeutics

[0123] The cDNAs and fragments thereof can be used in gene therapy.cDNAs can be delivered ex vivo to target cells, such as cells of bonemarrow. Once stable integration and transcription and or translation areconfirmed, the bone marrow may be reintroduced into the subject.Expression of the protein encoded by the cDNA may correct a disorderassociated with mutation of a normal sequence, reduction or loss of anendogenous target protein, or overepression of an endogenous or mutantprotein. Alternatively, cDNAs may be delivered in vivo using vectorssuch as retrovirus, adenovirus, adeno-associated virus, herpes simplexvirus, and bacterial plasmids. Non-viral methods of gene deliveryinclude cationic liposomes, polylysine conjugates, artificial viralenvelopes, and direct injection of DNA (Anderson (1998) Nature392:25-30; Dachs et al. (1997) Oncol Res 9:313-325; Chu et al. (1998) JMol Med 76(3-4):184-192; Weiss et al. (1999) Cell Mol Life Sci55(3):334-358; Agrawal (1996) Antisense Therapeutics, Humana Press,Totowa N.J.; and August et al. (1997) Gene Therapy (Advances inPharmacology, Vol. 40), Academic Press, San Diego Calif.).

[0124] In addition, expression of a particular protein can be regulatedthrough the specific binding of a fragment of a cDNA to a genomicsequence or an mRNA which encodes the protein or directs itstranscription or translation. The cDNA can be modified or derivatized toany RNA-like or DNA-like material including peptide nucleic acids,branched nucleic acids, and the like. These sequences can be producedbiologically by transforming an appropriate host cell with a vectorcontaining the sequence of interest.

[0125] Molecules which regulate the activity of the cDNA or encodedprotein are useful as therapeutics for diabetes mellitus, obesity,hypertension, atherosclerosis, polycystic ovarian syndrome, and cancersincluding breast, prostate, and colon. Such molecules include agonistswhich increase the expression or activity of the polynucleotide orencoded protein, respectively; or antagonists which decrease expressionor activity of the polynucleotide or encoded protein, respectively. Inone aspect, an antibody which specifically binds the protein may be useddirectly as an antagonist or indirectly as a delivery mechanism forbringing a pharmaceutical agent to cells or tissues which express theprotein.

[0126] Additionally, any of the proteins, or their ligands, orcomplementary nucleic acid sequences may be administered aspharmaceutical compositions or in combination with other appropriatetherapeutic agents. Selection of the appropriate agents for use incombination therapy may be made by one of ordinary skill in the art,according to conventional pharmaceutical principles. The combination oftherapeutic agents may act synergistically to affect the treatment orprevention of the conditions and disorders associated with an immuneresponse. Using this approach, one may be able to achieve therapeuticefficacy with lower dosages of each agent, thus reducing the potentialfor adverse side effects. Further, the therapeutic agents may becombined with pharmaceutically-acceptable carriers including excipientsand auxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Further details ontechniques for formulation and administration used by doctors andpharmacists may be found in the latest edition of Remington'sPharmaceutical Sciences (Mack Publishing, Easton Pa.).

[0127] Model Systems

[0128] Animal models may be used as bioassays where they exhibit aphenotypic response similar to that of humans and where exposureconditions are relevant to human exposures. Mammals are the most commonmodels, and most infectious agent, cancer, drug, and toxicity studiesare performed on rodents such as rats or mice because of low cost,availability, lifespan, reproductive potential, and abundant referenceliterature. Inbred and outbred rodent strains provide a convenient modelfor investigation of the physiological consequences of underexpressionor overexpression of genes of interest and for the development ofmethods for diagnosis and treatment of diseases. A mammal inbred tooverexpress a particular gene (for example, secreted in milk) may alsoserve as a convenient source of the protein expressed by that gene.

[0129] Transgenic Animal Models

[0130] Transgenic rodents that overexpress or underexpress a gene ofinterest may be inbred and used to model human diseases or to testtherapeutic or toxic agents. (See, e.g., U.S. Pat. No. 5,175,383 andU.S. Pat. No. 5,767,337.) In some cases, the introduced gene may beactivated at a specific time in a specific tissue type during fetal orpostnatal development. Expression of the transgene is monitored byanalysis of phenotype, of tissue-specific mRNA expression, or of serumand tissue protein levels in transgenic animals before, during, andafter challenge with experimental drug therapies.

[0131] Embryonic Stem Cells

[0132] Embryonic (ES) stem cells isolated from rodent embryos retain thepotential to form embryonic tissues. When ES cells such as the mouse129/SvJ cell line are placed in a blastocyst from the C57BL/6 mousestrain, they resume normal development and contribute to tissues of thelive-born animal. ES cells are preferred for use in the creation ofexperimental knockout and knockin animals. The method for this processis well known in the art and the steps are: the cDNA is introduced intoa vector, the vector is transformed into ES cells, transformed cells areidentified and microinjected into mouse cell blastocysts, blastocystsare surgically transferred to pseudopregnant dams. The resultingchimeric progeny are genotyped and bred to produce heterozygous orhomozygous strains.

[0133] Knockout Analysis

[0134] In gene knockout analysis, a region of a gene is enzymaticallymodified to include a non-natural intervening sequence such as theneomycin phosphotransferase gene (neo; Capecchi (1989) Science244:1288-1292). The modified gene is transformed into cultured ES cellsand integrates into the endogenous genome by homologous recombination.The inserted sequence disrupts transcription and translation of theendogenous gene.

[0135] Knockin Analysis

[0136] ES cells can be used to create knockin humanized animals ortransgenic animal models of human diseases. With knockin technology, aregion of a human gene is injected into animal ES cells, and the humansequence integrates into the animal cell genome. Transgenic progeny orinbred lines are studied and treated with potential pharmaceuticalagents to obtain information on the progression and treatment of theanalogous human condition.

[0137] As described herein, the uses of the cDNAs, provided in theSequence Listing of this application, and their encoded proteins areexemplary of known techniques and are not intended to reflect anylimitation on their use in any technique that would be known to theperson of average skill in the art. Furthermore, the cDNAs provided inthis application may be used in molecular biology techniques that havenot yet been developed, provided the new techniques rely on propertiesof nucleotide sequences that are currently known to the person ofordinary skill in the art, e.g., the triplet genetic code, specific basepair interactions, and the like. Likewise, reference to a method mayinclude combining more than one method for obtaining or assembling fulllength cDNA sequences that will be known to those skilled in the art. Itis also to be understood that this invention is not limited to theparticular methodology, protocols, and reagents described, as these mayvary. It is also understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims. The examples below are provided to illustratethe subject invention and are not included for the purpose of limitingthe invention.

EXAMPLES

[0138] It is to be understood that this invention is not limited to theparticular devices, machines, materials and methods described. Althoughparticular embodiments are described, equivalent embodiments may be usedto practice the invention. The described embodiments are provided toillustrate the invention and are not intended to limit the scope of theinvention which is limited only by the appended claims.

[0139] I cDNA Library Construction

[0140] The cDNA library SMCCNOS01 was selected as an example todemonstrate the construction of cDNA libraries from which the cDNAsco-expressed with known atherosclerosis-associated genes were derived.The SMCCNOS01 subtracted coronary artery smooth muscle cell library wasconstructed using 7.56×10⁶ clones from the SMCCNOT02 library and wassubjected to two rounds of subtraction hybridization for 48 hours with6.12×10⁶ clones from SMCCNOT01.

[0141] The SMCCNOT02 library was constructed using RNA isolated fromcoronary artery smooth muscle cells removed from a 3-year-old Caucasianmale. The cells were treated for 20 hours with TNFα and IL-1β at 10ng/ml each. The SMCCNOT01 was constructed using RNA isolated fromuntreated coronary artery smooth muscle cells from the same donor.Subtractive hybridization conditions were based on the methodologies ofSwaroop et al. (1991; Nucleic Acids Res 19:1954) and Bonaldo et al.(1996; Genome Research 6:791).

[0142] For both cDNA libraries, SMCCNOT01 and SMCCNOT02, the frozencoronary artery smooth muscle cells (50-100 mg) were homogenized in GTCbuffer (4.0M guanidine thiocyanate, 0.1M Tris-HCl pH 7.5, 1%2-mercaptoethanol). Two volumes of binding buffer (0.4M LiCl, 0.1MTris-HCl pH 7.5, 0.02M EDTA) were added, and the resulting mixture wasvortexed at 13,000 rpm. The supernatant was removed and combined witholigo d(T)₂₅ bound streptavidin particles (MPG). After rotation at roomtemperature, the mRNA-oligo d(T)₂₅ bound streptavidin particles wereseparated from the supernatant, washed twice with hybridization buffer 1(0.15M NaCl, 0.01M Tris-HCl pH 8.0, 1 mM EDTA, 0.1% lauryl sarcosinate)using magnetic separation at each step to remove the supernatant fromthe particles. Bound mRNA was eluted from the particles with releasesolution and heated to 65° C. The supernatant containing eluted mRNA wasmagnetically separated from the particles and used to construct the cDNAlibraries.

[0143] The RNA was used according to the recommended protocols in theSUPERSCRIPT plasmid system (Invitrogen). The cDNAs were fractionated ona SEPHAROSE CLIB column (APB), and those cDNAs exceeding 400 bp wereligated into pINCY plasmid (Incyte Genomics, Palo Alto Calif.).Recombinant plasmids were transformed into DH5α competent cells orELECTROMAX cells (Invitrogen).

[0144] II Isolation and Sequencing of cDNA Clones

[0145] Plasmid DNA was released from the cells and purified using theREAL Prep 96 plasmid kit (Qiagen, Valencia Calif.). The recommendedprotocol was employed except for the following changes: 1) the bacteriawere inoculated into 1 ml of sterile TERRIFIC BROTH (BD Biosciences, SanJose Calif.) with carbenicillin at 25 mg/l and glycerol at 0.4%; 2) thecells were cultured for 19 hours and then lysed with 0.3 ml of lysisbuffer; 3) following isopropanol precipitation, the plasmid DNA pelletwas resuspended in 0.1 ml distilled water, and 4) the samples weretransferred to a 96-well block for storage at 4° C.

[0146] The cDNAs were prepared using a MICROLAB 2200 system (Hamilton,Reno Nev.) in combination with the DNA ENGINE thermal cycler (MJResearch, Watertown Mass.). cDNAs were sequenced by the method of Sangeret al. (1975, J Mol Biol 94:441-446) using PRISM 377 (ABI) or MEGABACE1000 sequencing systems (APB).

[0147] Most of the sequences were sequenced using standard protocols andkits (ABI) at solution volumes of 0.25×-1.0×concentrations. In thealternative, some of the sequences disclosed herein were sequenced usingsolutions and dyes from APB.

[0148] III Selection, Assembly, and Characterization of Sequences

[0149] The sequences used for co-expression analysis were assembled fromEST sequences, 5′ and 3′ longread sequences, and full length codingsequences. The cDNAs claimed herein were expressed in at least threelibraries.

[0150] The assembly process is described as follows. EST sequencechromatograms were processed and verified. Quality scores were obtainedusing PHRED (Ewing et al. (1998) Genome Res 8:175-185; Ewing and Green(1998) Genome Res 8:186-194), and edited sequences were loaded into arelational database management system (RDBMS). The sequences wereclustered using BLAST with a product score of 50. All clusters of two ormore sequences created a bin which represents one transcribed gene.

[0151] Assembly of the component sequences within each bin was performedusing a modification of Phrap, a publicly available program forassembling DNA fragments (Green, P. University of Washington, SeattleWash.). Bins that showed 82% identity from a local pair-wise alignmentbetween any of the consensus sequences were merged.

[0152] Bins were annotated by screening the consensus sequence in eachbin against public databases, such as GBpri and GenPept from NCBI. Theannotation process involved a FASTn screen against the GBpri database inGenBank. Those hits with a percent identity of greater than or equal to75% and an alignment length of greater than or equal to 100 base pairswere recorded as homolog hits. The residual unannotated sequences werescreened by FASTx against GenPept. Those alignments with an E value ofless than or equal to 10⁻⁸ were recorded as homologs.

[0153] Sequences were then reclustered using BLASTn and Cross-Match, aprogram for rapid amino acid and nucleic acid sequence comparison anddatabase search (Green, supra), sequentially. Any BLAST alignmentbetween a sequence and a consensus sequence with a score greater than150 was realigned using cross-match. The sequence was added to the binwhose consensus sequence gave the highest Smith-Waterman score (Smith etal. (1992) Protein Engineering 5:35-51) amongst local alignments with atleast 82% identity. Non-matching sequences were moved into new bins, andassembly processes were repeated.

[0154] IV Coexpression Analyses of Atherosclerosis-Associated Genes

[0155] Known atherosclerosis-associated genes were selected to identifythe cDNAs that are closely associated with atherosclerosis. The knownatherosclerosis-associated genes which were used in this analysis alloccur within the LIFESEQ Gold database (Incyte Genomics), and briefdescriptions of their functions as they have been reported in theliterature are listed in Table 3. TABLE 3 Descriptions of KnownAtherosclerosis-Associated Genes GENE DESCRIPTION AND REFERENCES Human22kDa Smooth muscle cell-specific gene which is down-regulated duringsmooth smooth muscle muscle cell dedifferentiation as part ofatherogenic process (Sobue et al. (1998) protein (SM22) Horm Res50(S2):15-24; Sobue et al. (1999) Mol Cell Biochem 190:105-18) calponin(CNN1) Calponin is smooth muscle-specific and may mediate smooth musclecontractility through binding of the amino-terminal end of the myosinregulatory light chain. Involved in phenotypic modulation of smoothmuscle cells, a feature of atherosclerosis (Szymanski et al.(1999)Biochemistry 38:3778-84) pro alpha 1(I) Member of family of fibrousstructural proteins. Most abundant structural collagen (COL1A1)component of the extracellular matrix. Secreted as procollagen andconverted to collagen by matrix metalloproteinases. Collagens areimportant in atherosclerosis for promoting platelet aggregation and forproviding sites for platelet adhesion to the vessel wall (Wen et al.(1999) Arterioscler Thromb Vasc Biol 19:519-24) collagen alpha-2 typesee COL1A1 above I (COL1A2) COL6A1 see COL1A1 above procollagen alphasee COL1A1 above 2(V) (COL5A2) collagen VI alpha-2 see COL1A1 above(COL6A2) type VI collagen see COL1A1 above alpha3 (COL6A3) pro-alpha-1type 3 see COL1A1 above collagen (COL3A1) pro-alpha-1 (V) see COL1A1above collagen (COL3A1) matrix Gla protein Role in active calcificationof vascular smooth muscle cells, suggested by (MGP) expression study onVSMC in vitro differentiation study. Calcifying phenotype associatedwith high MGP levels. MGP knockout mice develop to term, but die up to 2months after birth due to extensive calcification of the arteries,causing blood vessel rupture (Luo et al. (1997) Nature 386:78-81; Moriet al. (1998) FEES Lett 433:19-22) cathepsin K (CTSK) Nonmetalloenzyme,potent elastase present in advanced atherosclerotic plaques. Contributesto the breakdown of components of vascular extracellular matrix,reducing tensile strength, increasing plaque vulnerability (Sukhova etal.(1998) J Clin Invest 102:576-83) fibrinogen beta chain Component offibrin in the extracellular matrix. Fibrin deposition is an integralgene (FGB) part of advanced atherosclerotic lesion development.Variation at the beta fibrinogen locus associated with peripheralatherosclerosis (Sueishi et al. (1998) Semin Thromb Hemost 24:255-260;Fowkes et al.(1992) Lancet 339:693-696) pre-pro-von Blood glycoproteininvolved in normal hemostasis. Mediates adhesion of Willebrand factorplatelets to sites of vascular damage. Also acts as a cofactor in factorVIII (VWF) activity in blood coagulation. Increased levels of VWF arefound in atherosclerosis and in several of its major risk factors,including hypercholesterolemia, diabetes, obesity, hypertension. Levelsserve as a predictor of adverse clinical outcome following vascularsurgery, possibly as an indicator of thrombus formation (Sadler (1998)Annu Rev Biochem 67:395-424 Blann et al. (1994) Eur J Vase Surg 8:10-15;Kessler et al. (1998) Diabetes Metab 24:327-36; Folsom et al. (1997)Circulation 96:1102-1108) platelet endothelial Signaling molecule in themigration of cells as part of the pathophysiology of cell adhesionvascular occulsive diseases such as atherosclerosis. Analysis ofmolecule endothelial/monocyte co-cultures indicates oxidative stressinduces (PECAM-1) transendothelial migration of monocytes as a result ofphosphorylation of PECAM-1 (Rattan et al. (1997) Am J Physiol273:E453-61) antithrombin III ATIII is the sole blood component throughwhich heparin exerts its anti- variant (AT3) coagulation effect.Deficiency in ATIII causes recurrent venous thrombosis and pulmonaryembolism and can be inherited in autosomal dominant fashion (Hultin etal. (1988) Thromb Haemost 59:468-73; Lane et al. (1996) Blood Rev10:59-74) lipoprotein lipase Hydrolyses triglyceride in chylomicrons andtherefore regulates metabolism of (LPL) circulating lipoproteins.Appears to have an atherogenic effect on the arterial wall due to itsability to alter the properties of LDL. Increased activity of LPL isfound in atherosclerotic arteries when compared to normal. Expressed bymacrophages in atherosclerotic lesions. Mutations in LPL responsible forfamilial hypercholesterolemia and premature atherosclerosis (Fisher etal. (1997) Atherosclerosis 135:145-159; Goldberg (1996) J Lipid Res37:693-707; Gerdes et al. (1997) Circulation 96:733-740) alpha-2-macro-Foam cell formation—retains LDL cholesterol in the lipid core ofglobulin (A2M) atherosclerotic plaque (Llorente et al. (1998) Rev EspCardiol 51:633-641) apolipoprotein AI Participates in reversecholesterol transport from tissues to the liver. Promotes (APOA1)cholesterol efflux from tissues and acts as a cofactor for lecithincholesterol acyltransferase (LCAT). Mutations in ApoA1 and ofApoAI/CIII/AIV gene cluster assoc with atherosclerosis. Transgenic miceexpressing high plasma APOAI levels are protected from fatty streakdevelopment with a high atherogenic diet (Gordon et al. (1989)Circulation 79:8-15; Rubin et al. (1991) Nature 353:265-7; Karathanasiset al. (1987) Proc Natl Acad Sci 84:7198- 7202) apolipoprotein AII Majorcomponent of HDL. Appears to have an opposite effect to that of (APOA2)APOAI, though exact function unknown. APOAII may have ability to convertHDL from an anti- to a pro-inflammatory particle, with paraoxonasehaving a role in this transformation process. Plasma APOAII levelssignificantly associated with plasma free fatty acid levels. Transgenicmice expressing varying levels of APOAII show increased atheroscleroticlesions than wt when fed an atherogenic diet. Possible interactionbetween diet/genotype and atherogenic potential (Escola-Gil et al.(1998) J Lipid Res 39:457-462; Warden et al. (1993) Proc Natl Acad Sci90:10886-10890) apolipoprotein B-100 Main apolipoprotein of chylomicronsand low density lipoproteins. Mutations in (APOB) APOB100 underlyfamilial defective apolipoprotein B-100 in which patients suffer frompremature atherosclerosis. Mutations result in defect in binding of LDLto LDL receptor, and accumulation of plasma LDL. High-expressing APOBtransgenic mice exibit elevated VLDL-LDL cholesterol and atherogeniclesions (Callow et al. (1995) J Clin Invest 96:1639-1646; Brasaemle etal.(1997) J Biol Chem 272:9378-9387) lipoprotein apoCII Role inlipoprotein metabolism. Cofactor in the activity of lipoprotein lipasethe (APOC2) enzyme that hydrolyzes triglycerides in plasma and transfersthe fatty acids to tissues. Mutations in APOC2 responsible forhyperlipoproteinemia 1B, similar to lipoprotein lipase deficiency (Coxet al. (1978) N Engl J Med 299:1421-1424; Arimoto et al. (1998) J LipidRes 39:143-151) pre-apolipoprotein Inhibits lipoprotein lipase andhepatic lipase, decreases uptake of lymph CIII (APOC3) chylomicrons byhepatic cells. APOA3 possibly delays breakdown of triglyceride richparticles. SstI RFLP in apoCIII is associated with plasma triglycerideand apoCIII levels and hyperlipidemic phenotypes (Henderson et al.(1987)Hum Genet 75:62-65) apolipoprotein apoC- APOC4 is a lipid-bindingprotein that has the potential to alter lipid metabolism. IV (APOC4)Human APOC4 transgenic mice are hypertriglyceridaemic compared to normalcontrols (Allan et al. (1996) J Lipid Res 37:1510-1518) macrophageMediates binding, internalisation and processing of negatively-chargedmacro- scavenger receptor molecules. Implicated in the pathologicaldeposition of cholesterol in arterial type I (MSR1) walls duringatherogenesis (Han et al. (1998) Hum Mol Genet 7:1039-1046) Humanantigen Acts as a scavenger receptor for oxidised LDL. Transientregulation under CD36 gene (CD36) control of M-CSF duringmonocyte-macrophage differentiation increases foam cell accumulation,Possible role in atherogenesis: increased M-CSF levels detected inatherosclerotic lesions in rabbits and humans (Huh et al. (1996) Blood87:2020-2028; Aitman et al. (1999) Nat Genet 21:76-83) serum amyloid PPlasma glycoprotein expressed in atherosclerotic lesions. Interacts withcomponent (SAP) lipoproteins in specific manner (Li et al. (1995)Arterioscler Thromb Vasc Biol 15:252-257; Li et al. (1998) BiochemBiophys Res Commun 244:249-252) carboxyl ester lipase CEL geneexpression increases in presence of oxidised and native LDL in gene(CEL) vitro. It is expressed in the vessel wall and in aortic extracts -may interact with cholesterol to modulate progression of atherosclerosis(Li et al. (1998) Biochem J 329:675-679) paraoxonase 1 Serum esteraseexclusively associated with high-density lipoproteins; it might (PON1)confer protection against coronary artery disease by destroying pro-inflammatory oxidized lipids in oxidized low-density lipoproteins. PON1gln192- to-arg polymorphism associated with coronary artery disease.Association between PON1 genetic variation and plasma LDL, HDL andnon-HDL and apoB levels in genetically isolated Alberta Hutteritepopulation. When fed on a high-fat, high-cholesterol diet, PON1-nullmice were more susceptible to atherosclerosis than wild-type (Serrato etal. (1995) J Clin Invest 96:3005-3008; Boright et al. (1998)Atherosclerosis 139:131-136; Shih et al. (1998) Nature 394:284-287)paraoxonase 2 Serum esterase exclusively associated with high-densitylipoproteins; it might (PON2) confer protection against coronary arterydisease by destroying pro- inflammatory oxidized lipids in oxidizedlow-density lipoproteins. Common polymorphism at codon 311 (cys-ser) inPON2 associated with CHD alone and synergistically with the 192polymorphism in PON1 in Asian Indians. Association between geneticvariation in PON2 and plasma cholesterol and apolipoprotein A1 ingenetically isolated Alberta Hutterite population (Sanghera et al.(1998) Am J Hum Genet 62:36-44; Boright supra) paraoxonase 3 Serumesterase exclusively associated with high-density lipoproteins; it might(PON3) confer protection against coronary artery disease by destroyingpro- inflammatory oxidized lipids in oxidized low-density lipoproteins.Other members PON2, 3 associated with CHD and cholesterol levels(Laplaud et al. (1998) Clin Chem Lab Med 36:431-441) perilipin (PLIN)Lipid storage droplets of steroidogenic cells are surrounded byperilipins, family of phosphorylated proteins encoded by a singlegene,detected in adipocytes and steroidogenic cells. Possible role in lipidmetabolism (Brasaemle et al. (1997) J Biol Chem 272:9378-9387)Prostaglandin D2 Catalyses conversion of PGH2 to PGD2, a prostaglandinimportant in smooth synthase (PTGDS) muscle contraction/relaxation andpotent inhibitor of platelet aggregation. Northern analysis shows strongspecific expression in heart. Immunocyto- chemical localization tomyocardial and atrio endocardial cells, and accumulates in end-stageatherosclerotic plaques. High plasma levels detected in severe anginapatients (Eguchi et al. (1997) Proc Natl Acad Sci 94:14689-14694)Annexin Inhibits phospholipase A2 activity and the production ofarachidonic acid, the II/lipocortinII(ANX2) precursor of theinflammatory mediators prostaglandins and leukotrienes. ANX2 is animportant anti-inflammatory molecule that binds plasminogen and t- PAand is suspected of having a role in atherogenesis. Binding ofplasminoger to ANX2 is specifically inhibited by the excess atherogenicLp(a) (Hajjar et al. (1998) J Investig Med 46:364-369) AnnexinI/lipocortin(ANX1) Inhibits phospholipase A2 activity and production ofarachidonic acid, the precursor of the inflammatory mediatorsprostaglandins and leukotrienes. ANXI is an important anti-inflammatorymolecule (Wallner et al. (1986) Nature 320:77-81) Secreted protein,Extracellular glycoprotein secreted by endothelial cells which has asuspected acidic and rich in role in calcification of atheroscleroticplaques. Interacts with PDGF-B cysteine (SPARC) containing dimers andinhibits binding to its receptors. Expression of SPARC and PDGF isminimal in most adult tissues, but is enhanced following injury andadvanced atherosclerotic lesions. Selective expression of SPARC causesrounding of adherent endothelial cells and influences extravasation ofmacromolecules (Raines et al. (1992) Proc Natl Acad Sci 89:1281-1285;Goldblum et al. (1994) Proc Natl Acad Sci 91:3448-3452)

[0156] From a total of 45,233 assembled gene sequences, 25 cDNAs wereidentified (SEQ ID NOs:1-25 of the Sequence Listing) that show strongassociation with the known atherosclerosis-associated genes.

[0157] Initially, the degree of association was measured by probabilityvalues using a cutoff p-value less than 0.00001. The sequences werefurther examined to ensure that the genes that passed the probabilitytest had strong association with known atherosclerosis-associated genes.Details of the co-expression patterns for the known genes andco-expressed cDNAs are presented in Table 4. The entries in Table 4 arethe negative log of the p-value (−log p) for the coexpression of the twogenes. Table 5 summarizes the highly significant co-expressionrelationships between each cDNA, two marker genes and their functions.

[0158] V Atherosclerosis-Associated cDNAs

[0159] Using the co-expression analysis method, cDNAs comprising thepolynucleotides of SEQ ID NOs:1-25 and their complements, wereidentified by their highly significant co-expression with knownatherosclerosis-associated genes.

[0160] BLAST and other motif searches were performed for SEQ ID NOs:1-25according to Example VII. SEQ ID NO:8 was determined to be full lengthand translated as shown in FIG. 1.

[0161] VI Transcript Imaging

[0162] Transcript images were performed for several of the cDNAs of theinvention using the LIFESEQ GOLD database (July 02 release, IncyteGenomics). This process allowed assessment of the relative abundance ofthe expressed polynucleotides in all of the cDNA libraries, but those inthe cardiovascular category are specifically emphasized. Criteria fortranscript imaging can be selected from category, number of cDNAs perlibrary, library description, disease indication, clinical relevance ofsample, and the like.

[0163] All sequences and cDNA libraries in the LIFESEQ database havebeen categorized by system, organ/tissue and cell type. For eachcategory, the number of libraries in which the sequence was expressedwere counted and shown over the total number of libraries in thatcategory. For each library, the number of cDNAs were counted and shownover the total number of cDNAs in that library. In some transcriptimages, all normalized or subtracted libraries, which have high copynumber sequences removed prior to processing, and all mixed or pooledtissues, which are considered non-specific in that they contain morethan one tissue type or more than one subject's tissue, can be excludedfrom the analysis. Treated and untreated cell lines and/or fetal tissuedata can also be excluded where clinical relevance is emphasized.Conversely, fetal tissue can be emphasized wherever elucidation ofinherited disorders or differentiation of particular adult or embryonicstem cells into tissues or organs such as heart, kidney, nerves orpancreas would be aided by removing clinical samples from the analysis.Transcript imaging can also be used to support data from othermethodologies such as guilt-by-association and hybridization analyses.

[0164] The transcript images for SEQ ID NOs:3-6, 8,13, 15-20, and 22 areshown in Table 6. The first column shows library name; the secondcolumn, the number of cDNAs sequenced in that library; the third column,the description of the library; the fourth column, absolute abundance ofthe transcript in the library; and the fifth column, percentageabundance of the transcript in the library. In some cases, the normallibrary shows differential expression of the cDNA; in others, theinduced or diseased library shows differential expression. For example,for SEQ ID NO:8, the endothelial cells treated with growth factors (VEGFand EF) show 2-8×higher expression than untreated endothelial cells inthe same experiment.

[0165] These data confirm the differential expression of SEQ ID NOs:3-6,8,13, 15-20, and 22 under conditions that correlate with disordersassociated with atherosclerosis. VII Homology Searching forAtherosclerosis-Associated cDNAs and Polypeptides The polynucleotidesequences, SEQ ID NO:1-25, and polypeptide sequence, SEQ ID NO:26, werequeried against databases derived from sources such as GenBank andSwissProt. These databases, which contain previously identified andannotated sequences, were searched for regions of similarity using BLAST(Altschul, supra). BLAST searched for matches and reported only thosethat satisfied the probability thresholds of 10-25 or less fornucleotide sequences and 10-8 or less for polypeptide sequences.

[0166] The polypeptide sequence was also analyzed for known motifpatterns using MOTIFS, SPSCAN, BLIMPS, and HMM-based protocols. MOTIFS(Genetics Computer Group, Madison Wis.) searches polypeptide sequencesfor patterns that match those defined in the Prosite Dictionary ofProtein Sites and Patterns (Bairoch, supra) and displays the patternsfound and their corresponding literature abstracts. SPSCAN (GeneticsComputer Group) searches for potential signal peptide sequences using aweighted matrix method (Nielsen et al. (1997) Protein Engineering10:1-6). Hits with a score of 5 or greater were considered. BLIMPS usesa weighted matrix analysis algorithm to search for sequence similaritybetween the polypeptide sequences and those contained in BLOCKS, adatabase consisting of short amino acid segments, or blocks of 3-60amino acids in length, compiled from the PROSITE database (Henikoff;supra; Bairoch, supra), and those in PRINTS, a protein fingerprintdatabase based on non-redundant sequences obtained from sources such asSwissProt, GenBank, PIR, and NRL-3D (Attwood et al. (1997) J Chem InfComput Sci 37:417-424). For the purposes of the present invention, theBLIMPS searches reported matches with a cutoff score of 1000 or greaterand a cutoff probability value of 1.0×10⁻³. HMM-based protocols werebased on a probabilistic approach and searched for consensus primarystructures of gene families in the protein sequences (Eddy, supra;Sonnhammer, supra). More than 500 known protein families with cutoffscores ranging from 10 to 50 bits were selected for use in thisinvention.

[0167] VIII Hybridization Technologies: Selection of Sequences,Microarray Preparation and Use

[0168] SEQ ID NO:1-25 are represented among the template sequences inthe LIFESEQ GOLD database (Incyte Genomics). Several of these sequences,specifically SEQ ID NOs:1, 2, 10, 12, 18, and 32-34 have been used onmicroarrays in experiments investigating differential gene expression incardiovascular samples. Table 7 presents the results of theseexperiments; results were significant if the log2 Cy/Cy5 ratio exceeded±1.00 in either the normal or the induced or disease state.

[0169] Exemplary Experimental Materials and Protocols

[0170] Activation of the vascular endothelium is considered to be acentral event in a wide range of both physiological andpathophysiological processes, such as vascular tone regulation,coagulation and thrombosis, atherosclerosis, and inflammation.

[0171] HAEC, human aortic endothelial cells, are primary cells derivedfrom the endothelium of a human aorta. They have been used as anexperimental model for investigating the role of the endothelium inhuman vascular biology in vitro. HAECs were grown to 85% confluency,split into two samples, one of which was then treated with growthfactors, LDL, cytokines, O2, and the like for variable time periods.

[0172] ECV304, HUAEC, and HUVEC are endothelial cell lines derived fromthe endothelium of the human umbilical artery or vein. This cell modelhas been extensively used to study the functional biology of humanendothelial cells. These cells were also grown to about 85% confluency,split into samples, one of which was then treated with growth factors,LDL, cytokines, O2, and the like for variable time periods.

[0173] The experimental treatments and time of exposure are shown by thetissue description in Table 7.

[0174] Exemplary Activators and Inducers

[0175] TNF-α is a pleiotropic cytokine that is known to play a centralrole in the mediation of inflammatory responses through activation ofmultiple signal transduction pathways. TNF-α is produced by activatedlymphocytes, macrophages, and other white blood cells, and is known toactivate endothelial cells. Monitoring the endothelial cells' responseto TNF-α at the level of the mRNA expression can provide informationnecessary for better understanding of both TNF-α signaling pathways andendothelial cell biology.

[0176] PMA is a broad activator of the protein kinase C-dependentpathways. lonomycin is a calcium ionophore that permits the entry ofcalcium in the cell, hence increasing the cytosolic calciumconcentration. The combination of PMA and ionomycin activates two of themajor signaling pathways used by mammalian cells to interact with theirenvironment. In T cells, the combination of PMA and ionomycin mimics thetype of secondary signaling events elicited during optimal B cellactivation.

[0177] Microarrays

[0178] The HUMAN GENOME GEM series 1-5 microarrays (Incyte Genomics)contain 45,320 array elements which represent 22,632 annotated clustersand 22,688 unannotated clusters. For the UNIGEM series microarrays(Incyte Genomics), Incyte clones were mapped to non-redundant Unigeneclusters (Unigene database (build 46), NCBI; Shuler (1997) J Mol Med75:694-698), and the 5′ clone with the strongest BLAST alignment (atleast 90% identity and 100 bp overlap) was chosen, verified, and used inthe construction of the microarray. The UNIGEM V 2.0 microarray (IncyteGenomics) contains 8,502 array elements which represent 8,372 annotatedgenes and 130 unannotated clusters.

[0179] To construct microarrays, cDNAs were amplified from bacterialcells using primers complementary to vector sequences flanking the cDNAinsert. Thirty cycles of PCR increased the initial quantity of cDNAsfrom 1-2 ng to a final quantity greater than 5 μg. Amplified cDNAs werethen purified using SEPHACRYL-400 columns (APB). Purified cDNAs wereimmobilized on polymer-coated glass slides. Glass microscope slides(Corning, Corning N.Y.) were cleaned by ultrasound in 0.1% SDS andacetone, with extensive distilled water washes between and aftertreatments. Glass slides were etched in 4% hydrofluoric acid (VWRScientific Products, West Chester Pa.), washed thoroughly in distilledwater, and coated with 0.05% aminopropyl silane (Sigma-Aldrich) in 95%ethanol. Coated slides were cured in a 110° C. oven. cDNAs were appliedto the coated glass substrate using a procedure described in U.S. Pat.No. 5,807,522. One microliter of the cDNA at an average concentration of100 ng/μl was loaded into the open capillary printing element by ahigh-speed robotic apparatus which then deposited about 5 nl of cDNA perslide.

[0180] Microarrays were UV-crosslinked using a STRATALINKERUV-crosslinker (Stratagene), and then washed at room temperature once in0.2% SDS and three times in distilled water. Non-specific binding siteswere blocked by incubation of microarrays in 0.2% casein in phosphatebuffered saline (Tropix, Bedford Mass.) for 30 minutes at 60° C.followed by washes in 0.2% SDS and distilled water as before.

[0181] Isolation and Labeling of Sample cDNAs

[0182] Cells were harvested and lysed in 1 ml of TRIZOL reagent (5×10⁶cells/ml; Invitrogen). The lysates were vortexed thoroughly andincubated at room temperature for 2-3 minutes and extracted with 0.5 mlchloroform. The extract was mixed, incubated at room temperature for 5minutes, and centrifuged at 16,000×g for 15 minutes at 4° C. The aqueouslayer was collected, and an equal volume of isopropanol was added.Samples were mixed, incubated at room temperature for 10 minutes, andcentrifuged at 16,000×g for 20 minutes at 4° C. The supernatant wasremoved, and the RNA pellet was washed with 1 ml of 70% ethanol,centrifuged at 16,000×g at 4° C., and resuspended in RNAse-free water.The concentration of the RNA was determined by measuring the opticaldensity at 260 nm.

[0183] Poly(A) RNA was prepared using an OLIGOTEX mRNA kit (Qiagen) withthe following modifications: OLIGOTEX beads were washed in tubes ratherthan spin columns, resuspended in elution buffer, and then loaded ontospin columns to recover the mRNA. To obtain maximum yield, the mRNA waseluted twice.

[0184] Each poly(A) RNA sample was reverse transcribed using MMLVreverse-transcriptase, 0.05 pg/μl oligo-d(T) primer (21mer), 1×firststrand buffer, 0.03 units/ul RNAse inhibitor, 500 uM dATP, 500 uM dGTP,500 uM dTTP, 40 uM dCTP, and 40 uM either dCTP-Cy3 or dCTP-Cy5 (APB).The reverse transcription reaction was performed in a 25 ml volumecontaining 200 ng poly(A) RNA using the GEMBRIGHT kit (Incyte Genomics).Specific control poly(A) RNAs (YCFRO6, YCFR45, YCFR67, YCFR85, YCFR43,YCFR22, YCFR23, YCFR25, YCFR44, YCFR26) were synthesized by in vitrotranscription from non-coding yeast genomic DNA (W. Lei, unpublished).As quantitative controls, control mRNAs (YCFRO6, YCFR45, YCFR67, andYCFR85) at 0.002 ng, 0.02 ng, 0.2 ng, and 2 ng were diluted into reversetranscription reaction at ratios of 1:100,000, 1:10,000, 1:1000, 1:100(w/w) to sample mRNA, respectively. To sample differential expressionpatterns, control mRNAs (YCFR43, YCFR22, YCFR23, YCFR25, YCFR44, YCFR26)were diluted into reverse transcription reaction at ratios of 1:3, 3:1,1:10, 10:1, 1:25, 25:1 (w/w) to sample mRNA. Reactions were incubated at37° C. for 2 hr, treated with 2.5 ml of 0.5M sodium hydroxide, andincubated for 20 minutes at 85° C. to the stop the reaction and degradethe RNA.

[0185] cDNAs were purified using two successive CHROMA SPIN 30 gelfiltration spin columns (Clontech). Cy3- and Cy5-labeled reactionsamples were combined as described below and ethanol precipitated using1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%ethanol. The cDNAs were then dried to completion using a SpeedVAC system(Savant Instruments, Holbrook N.Y.) and resuspended in 14 μl 5×SSC, 0.2%SDS.

[0186] Hybridization and Detection

[0187] Hybridization reactions contained 9 μl of sample mixturecontaining 0.2 μg each of Cy3 and Cy5 labeled cDNA synthesis products in5×SSC, 0.2% SDS hybridization buffer. The mixture was heated to 65° C.for 5 minutes and was aliquoted onto the microarray surface and coveredwith an 1.8 cm² coverslip. The microarrays were transferred to awaterproof chamber having a cavity just slightly larger than amicroscope slide. The chamber was kept at 100% humidity internally bythe addition of 140 μl of 5×SSC in a corner of the chamber. The chambercontaining the microarrays was incubated for about 6.5 hours at 60° C.The microarrays were washed for 10 min at 45° C. in low stringency washbuffer (1×SSC, 0.1% SDS), three times for 10 minutes each at 45° C. inhigh stringency wash buffer (0.1×SSC), and dried.

[0188] Reporter-labeled hybridization complexes were detected with amicroscope equipped with an Innova 70 mixed gas 10 W laser (Coherent,Santa Clara Calif.) capable of generating spectral lines at 488 nm forexcitation of Cy3 and at 632 nm for excitation of Cy5. The excitationlaser light was focused on the microarray using a 20×microscopeobjective (Nikon, Melville N.Y.). The slide containing the microarraywas placed on a computer-controlled X-Y stage on the microscope andraster-scanned past the objective. The 1.8 cm×1.8 cm microarray used inthe present example was scanned with a resolution of 20 micrometers.

[0189] In two separate scans, the mixed gas multiline laser excited thetwo fluorophores sequentially. Emitted light was split, based onwavelength, into two photomultiplier tube detectors (PMT R1477;Hamamatsu Photonics Systems, Bridgewater N.J.) corresponding to the twofluorophores. Appropriate filters positioned between the microarray andthe photomultiplier tubes were used to filter the signals. The emissionmaxima of the fluorophores used were 565 nm for Cy3 and 650 nm for Cy5.Each microarray was typically scanned twice, one scan per fluorophoreusing the appropriate filters at the laser source, although theapparatus was capable of recording the spectra from both fluorophoressimultaneously.

[0190] The sensitivity of the scans was calibrated using the signalintensity generated by a cDNA control species. Samples of thecalibrating cDNA were separately labeled with the two fluorophores andidentical amounts of each were added to the hybridization mixture. Aspecific location on the microarray contained a complementary DNAsequence, allowing the intensity of the signal at that location to becorrelated with a weight ratio of hybridizing species of 1:100,000.

[0191] The output of the photomultiplier tube was digitized using a12-bit RTI-835H analog-to-digital (A/D) conversion board (AnalogDevices, Norwood, Mass.) installed in an IBM-compatible PC computer. Thedigitized data were displayed as an image where the signal intensity wasmapped using a linear 20-color transformation to a pseudocolor scaleranging from blue (low signal) to red (high signal). The data was alsoanalyzed quantitatively. Where two different fluorophores were excitedand measured simultaneously, the data were first corrected for opticalcrosstalk (due to overlapping emission spectra) between the fluorophoresusing each fluorophore's emission spectrum.

[0192] A grid was superimposed over the fluorescence signal image suchthat the signal from each spot was centered in each element of the grid.The fluorescence signal within each element was then integrated toobtain a numerical value corresponding to the average intensity of thesignal. The software used for signal analysis was the GEMTOOLS geneexpression analysis program (Incyte Genomics). Significance was definedas signal to background ratio exceeding 2×and area hybridizationexceeding 40%.

[0193] IX Further Characterization of Differentially Expressed cDNAs andProteins

[0194] Clones were aligned against the LIFESEQ Gold 5.1 database (IncyteGenomics) and an Incyte template and its sequence variants were chosenfor each clone. The template and variant sequences were aligned againstthe GenBank nucleotide sequence databases using BLASTn (vers. 2.0, NCBI)to acquire annotation. The template and variant sequences weretranslated into amino acid sequences which were aligned against GenPeptand other protein databases using BLASTp (vers. 2.0, NCBI) to acquireannotation and characterization, i.e., structural motifs. Table 3 showsthe GenBank annotations (where available) for SEQ ID NOs:1-25 of thisinvention as produced by BLAST analysis.

[0195] Percent sequence identity can be determined electronically fortwo or more amino acid or nucleic acid sequences using the MEGALIGNprogram, a component of LASERGENE software (DNASTAR). The percentidentity between two amino acid sequences is calculated by dividing thelength of sequence A, minus the number of gap residues in sequence A,minus the number of gap residues in sequence B, into the sum of theresidue matches between sequence A and sequence B, times one hundred.Gaps of low or of no homology between the two amino acid sequences arenot included in determining percentage identity.

[0196] Sequences with conserved protein motifs may be searched using theBLOCKS search program. This program analyses sequence informationcontained in the Swiss-Prot and PROSITE databases and is useful fordetermining the classification of uncharacterized proteins translatedfrom genomic or cDNA sequences (Bairoch, supra; Attwood, supra). PROSITEdatabase is a useful source for identifying functional or structuraldomains that are not detected using motifs due to extreme sequencedivergence. Using weight matrices, these domains are calibrated againstthe SWISS-PROT database to obtain a measure of the chance distributionof the matches.

[0197] The PRINTS database can be searched using the BLIMPS searchprogram to obtain protein family “fingerprints”. The PRINTS databasecomplements the PROSITE database by exploiting groups of conservedmotifs within sequence alignments to build characteristic signatures ofdifferent protein families. For both BLOCKS and PRINTS analyses, thecutoff scores for local similarity were:

[0198] >1300=strong, 1000-1300=suggestive; for global similarity were:p<exp−3; and for strength (degree of correlation) were: >1300=strong,1000-1300=weak.

[0199] X Other Hybridization Technologies and Analyses

[0200] Other hybridization technologies utilize a variety of substratessuch as nylon membranes, capillary tubes, etc. Arranging cDNAs onpolymer coated slides is described in Example V; sample cDNA preparationand hybridization and analysis using polymer coated slides is describedin examples VI and VII, respectively.

[0201] The cDNAs are applied to a membrane substrate by one of thefollowing methods. A mixture of cDNAs is fractionated by gelelectrophoresis and transferred to a nylon membrane by capillarytransfer. Alternatively, the cDNAs are individually ligated to a vectorand inserted into bacterial host cells to form a library. The cDNAs arethen arranged on a substrate by one of the following methods. In thefirst method, bacterial cells containing individual clones arerobotically picked and arranged on a nylon membrane. The membrane isplaced on LB agar containing selective agent (carbenicillin, kanamycin,ampicillin, or chloramphenicol depending on the vector used) andincubated at 37° C. for 16 hr. The membrane is removed from the agar andconsecutively placed colony side up in 10% SDS, denaturing solution (1.5M NaCl, 0.5 M NaOH), neutralizing solution (1.5 M NaCl, 1 M Tris, pH8.0), and twice in 2×SSC for 10 min each. The membrane is then UVirradiated in a STRATALINKER UV-crosslinker (Stratagene).

[0202] In the second method, cDNAs are amplified from bacterial vectorsby thirty cycles of PCR using primers complementary to vector sequencesflanking the insert. PCR amplification increases a startingconcentration of 1-2 ng nucleic acid to a final quantity greater than 5μg. Amplified nucleic acids from about 400 bp to about 5000 bp in lengthare purified using SEPHACRYL-400 beads (APB). Purified nucleic acids arearranged on a nylon membrane manually or using a dot/slot blottingmanifold and suction device and are immobilized by denaturation,neutralization, and UV irradiation as described above.

[0203] Hybridization probes derived from cDNAs of the Sequence Listingare employed for screening cDNAs, mRNAs, or genomic DNA inmembrane-based hybridizations. Probes are prepared by diluting the cDNAsto a concentration of 40-50 ng in 45 IL TE buffer, denaturing by heatingto 100° C. for five min and briefly centrifuging. The denatured cDNA isthen added to a REDIPRIME tube (APB), gently mixed until blue color isevenly distributed, and briefly centrifuged. Five microliters of[³²P]dCTP is added to the tube, and the contents are incubated at 37° C.for 10 min. The labeling reaction is stopped by adding 5 μl of 0.2MEDTA, and probe is purified from unincorporated nucleotides using aPROBEQUANT G-50 microcolumn (APB). The purified probe is heated to 100°C. for five min and then snap cooled for two min on ice.

[0204] Membranes are pre-hybridized in hybridization solution containing1% Sarkosyl and Ix high phosphate buffer (0.5 M NaCl, 0.1 M Na₂HPO₄, 5mM EDTA, pH 7) at 55° C. for two hr. The probe, diluted in 15 ml freshhybridization solution, is then added to the membrane. The membrane ishybridized with the probe at 55° C. for 16 hr. Following hybridization,the membrane is washed for 15 min at 25° C. in imM Tris (pH 8.0), 1%Sarkosyl, and four times for 15 min each at 25° C. in 1 mM Tris (pH8.0). To detect hybridization complexes, XOMAT-AR film (Eastman Kodak,Rochester N.Y.) is exposed to the membrane overnight at −70° C.,developed, and examined.

[0205] XI Expression of the Encoded Protein

[0206] Expression and purification of a protein encoded by a cDNA of theinvention is achieved using bacterial or virus-based expression systems.For expression in bacteria, cDNA is subcloned into a vector containingan antibiotic resistance gene and an inducible promoter that directshigh levels of cDNA transcription. Examples of such promoters include,but are not limited to, the trp-lac (tac) hybrid promoter and the T5 orT7 bacteriophage promoter in conjunction with the lac operatorregulatory element. Recombinant vectors are transformed into bacterialhosts, such as BL21(DE3). Antibiotic resistant bacteria express theprotein upon induction with IPTG. Expression in eukaryotic cells isachieved by infecting Spodoptera frugiperda (Sf9) insect cells withrecombinant baculovirus, Autographica californica nuclear polyhedrosisvirus. The polyhedrin gene of baculovirus is replaced with the cDNA byeither homologous recombination or bacterial-mediated transpositioninvolving transfer plasmid intermediates. Viral infectivity ismaintained and the strong polyhedrin promoter drives high levels oftranscription.

[0207] For ease of purification, the protein is synthesized as a fusionprotein with glutathione-S-transferase (GST; APB) or a similaralternative such as FLAG. The fusion protein is purified on immobilizedglutathione under conditions that maintain protein activity andantigenicity. After purification, the GST moiety is proteolyticallycleaved from the protein with thrombin. A fusion protein with FLAG, an8-amino acid peptide, is purified using commercially availablemonoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak, RochesterN.Y.).

[0208] XII Production of Antibodies

[0209] A denatured protein from a reverse phase HPLC separation isobtained in quantities up to 75 mg. This denatured protein is used toimmunize mice or rabbits following standard protocols. About 100 μg isused to immunize a mouse, while up to 1 mg is used to immunize a rabbit.The denatured protein is radioiodinated and incubated with murine B-cellhybridomas to screen for monoclonal antibodies. About 20 mg of proteinis sufficient for labeling and screening several thousand clones.

[0210] In another approach, the amino acid sequence translated from acDNA of the invention is analyzed using PROTEAN software (DNASTAR) todetermine antigenic determinants of the protein. The optimal sequencesfor immunization are usually at the C-terminus, the N-terminus, andthose intervening, hydrophilic regions of the protein that are likely tobe exposed to the external environment when the protein is in itsnatural conformation. Typically, oligopeptides about 15 residues inlength are synthesized using an 431 peptide synthesizer (ABI) usingFmoc-chemistry and then coupled to keyhole limpet hemocyanin (KLH;Sigma-Aldrich) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimideester. If necessary, a cysteine may be introduced at the N-terminus ofthe peptide to permit coupling to KLH. Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity by binding the peptide toplastic, blocking with 1% BSA, reacting with rabbit antisera, washing,and reacting with radioiodinated goat anti-rabbit IgG.

[0211] Hybridomas are prepared and screened using standard techniques.Hybridomas of interest are detected by screening with radioiodinatedprotein to identify those fusions producing a monoclonal antibodyspecific for the protein. In a typical protocol, wells of 96 well plates(FAST, Becton-Dickinson, Palo Alto Calif.) are coated withaffinity-purified, specific rabbit-anti-mouse (or suitable anti-speciesIg) antibodies at 10 mg/ml. The coated wells are blocked with 1% BSA andwashed and exposed to supernatants from hybridomas. After incubation,the wells are exposed to radiolabeled protein at 1 mg/ml. Clonesproducing antibodies bind a quantity of labeled protein that isdetectable above background.

[0212] Such clones are expanded and subjected to 2 cycles of cloning at1 cell/3 wells. Cloned hybridomas are injected into pristane-treatedmice to produce ascites, and monoclonal antibody is purified from theascitic fluid by affinity chromatography on protein A (APB). Monoclonalantibodies with affinities of at least 10⁸ M⁻, preferably 10⁹ to 1010M⁻¹ or stronger, are made by procedures well known in the art.

[0213] XIII Purification of Naturally Occurring Protein Using Antibodies

[0214] Naturally occurring or recombinant protein is purified byimmunoaffinity chromatography using antibodies specific for the protein.An immunoaffinity column is constructed by covalently coupling theantibody to CNBr-activated SEPHAROSE resin (APB). Media containing theprotein is passed over the immunoaffinity column, and the column iswashed using high ionic strength buffers in the presence of detergent toallow preferential absorbance of the protein. After coupling, theprotein is eluted from the column using a buffer of pH 2-3 or a highconcentration of urea or thiocyanate ion to disrupt antibody/proteinbinding, and the protein is collected.

[0215] XIV Screening for Molecules That Specifically Bind the cDNA orProtein

[0216] The cDNA or fragments thereof and the protein or portions thereofare labeled with ³²P-dCTP, Cy3-dCTP, Cy5-dCTP (APB), or BIODIPY or FITC(Molecular Probes), respectively. Candidate molecules or compoundspreviously arranged on a substrate are incubated in the presence oflabeled nucleic or amino acid. After incubation under conditions foreither a cDNA or a protein, the substrate is washed, and any position onthe substrate retaining label, which indicates specific binding orcomplex formation, is assayed. The binding molecule is identified by itsarrayed position on the substrate. Data obtained using differentconcentrations of the nucleic acid or protein are used to calculateaffinity between the labeled nucleic acid or protein and the boundmolecule. High throughput screening using very small assay volumes andvery small amounts of test compound is fully described in U.S. Pat. No.5,876,946.

[0217] All patents and publications mentioned in the specification areincorporated herein by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in thefield of molecular biology or related fields are intended to be withinthe scope of the following claims. TABLE 4 Co-expression of 25 cDNAswith known atherosclerosis genes (- log p). SEQ ID CNN1 COL1A1 COL1A2COL6A1 COL5A2 COL6A2 COL6A3 COL3A1 COL5A1 MGP CTSK FGB VWF 1 0 1 0 0 0 00 0 0 0 0 4 1 2 1 1 3 2 2 2 4 3 4 5 3 1 6 3 0 1 0 1 1 1 0 0 1 1 1 4 0 41 0 1 1 1 0 1 1 1 0 1 7 0 5 5 9 12 13 3 14 11 12 4 10 13 1 3 6 6 4 7 9 413 5 6 8 14 6 1 12 7 5 4 5 5 2 7 5 6 5 10 1 2 1 8 1 1 0 2 1 1 0 1 0 1 01 1 9 1 1 4 2 1 6 4 4 5 6 1 0 10 10 3 4 3 3 3 4 4 3 1 4 1 0 5 11 1 1 1 11 1 1 0 1 1 2 1 1 12 11 4 7 9 2 9 11 10 7 11 3 0 5 13 2 6 5 6 9 8 4 6 71 3 1 1 14 0 0 0 0 0 0 0 1 0 1 1 4 1 15 17 7 13 17 2 25 12 11 6 21 6 3 716 13 22 27 27 15 33 24 34 15 20 18 0 8 17 2 1 3 3 0 2 6 3 1 9 1 0 7 186 5 6 7 3 7 10 6 2 14 5 0 5 19 0 1 1 2 1 1 0 1 0 0 1 7 0 20 8 7 8 12 3 911 10 3 11 6 1 8 21 0 2 4 2 4 2 5 5 3 2 1 0 4 22 1 1 0 1 3 1 2 1 1 1 1 41 23 1 1 0 1 0 1 0 1 0 0 0 7 0 24 0 1 2 0 0 0 0 1 0 1 0 8 1 25 48 11 1724 3 26 18 17 13 20 6 1 8 SEQ ID PECAM1 AT3 LPL A2M APOA1 APOA2 APOBAPOC2 APOC3 APOC4 MSR1 CD36 SAP CEL 1 0 5 0 1 3 4 2 2 5 4 0 0 6 0 2 7 01 4 0 1 0 0 0 0 2 3 0 1 3 0 8 1 1 5 4 3 3 5 7 0 0 8 1 4 0 4 1 2 4 5 4 46 2 0 1 3 0 5 2 0 4 9 1 0 0 1 0 0 4 2 0 2 6 6 1 5 7 0 1 1 0 1 0 7 3 0 27 4 3 3 8 1 1 2 0 2 1 2 7 2 0 8 0 0 3 3 0 0 0 2 0 0 1 1 1 0 9 8 0 5 6 11 1 2 0 0 5 6 0 1 10 3 0 4 4 0 0 0 0 0 0 9 5 0 0 11 1 2 1 0 3 1 2 2 1 00 2 3 6 12 2 1 3 6 2 0 1 0 0 0 4 3 0 3 13 0 0 1 1 3 2 1 2 1 0 1 2 1 3 140 5 1 1 4 5 5 5 3 1 0 0 2 1 15 5 1 1 13 1 1 1 0 1 0 10 1 1 0 16 7 0 3 121 0 0 1 1 0 9 5 0 1 17 5 0 6 7 0 0 0 1 0 0 5 9 0 1 18 4 0 6 10 1 0 1 0 00 8 5 1 1 19 1 8 1 3 5 8 4 6 8 9 1 1 11 0 20 2 0 12 10 1 1 0 1 0 0 9 140 0 21 4 0 8 5 0 1 0 1 1 0 5 11 1 1 22 4 9 0 2 6 8 7 6 4 3 1 1 7 0 23 06 0 1 6 7 7 4 8 1 0 1 7 1 24 0 7 0 2 8 10 10 7 8 4 0 0 5 1 25 2 0 2 20 10 3 0 0 0 10 4 0 1 SEQ ID PON1 PON2 PON3 PLIN PTGDS ANX2 ANX1 SPARC SM221 5 1 2 0 0 2 0 1 0 2 1 6 1 1 2 3 4 8 3 3 5 2 2 0 0 1 0 1 0 4 2 0 1 0 01 0 1 1 5 1 1 1 6 4 6 3 9 9 6 1 4 0 11 7 6 4 11 14 7 1 4 5 2 1 11 7 7 88 1 5 0 1 23 1 1 4 2 9 0 7 1 6 3 3 2 7 4 10 0 1 0 15 2 2 1 2 4 11 6 2 74 0 0 2 1 0 12 1 3 1 3 3 6 3 7 9 13 2 3 2 2 1 1 3 4 2 14 2 2 1 1 0 0 0 10 15 0 2 1 1 12 7 9 14 27 16 1 2 2 7 6 12 9 22 20 17 0 1 0 6 2 3 1 6 318 1 2 3 8 6 3 10 6 11 19 5 1 2 0 1 0 1 0 1 20 0 0 0 19 4 4 2 7 10 21 01 0 8 0 2 2 3 3 22 4 2 2 1 2 1 3 1 1 23 4 1 3 1 0 0 0 1 0 24 3 1 1 0 2 11 1 0 25 1 1 1 3 11 9 6 9 30

[0218] TABLE 5 Summary of coexpressed genes and their function inatherosclerosis-associated disorders SEQ ID Pvalue Gene 1 Gene functionfrom Table 3 Pvalue Gene 2 Gene function from Table 3 1 6 SAP associatedwith amyloid P 5 APOC3 associated with plasma in lesions triglycerideand hyperlipidemia 2 8 SPARC calcification of plaques 7 PECAM-implicated in migration of cells under O2 stress 3 8 AT3 deficiencycauses recurrent 8 SAP associated with amyloid P in venous thrombosislesions 4 7 FGB fibrin deposition in plaque 6 APOC3 apolipoprotein CIII5 14 COL6A2 promoting platelet aggregation 12 CTSK present in advancedplaques 6 14 SM22 downregulated during 14 MGP calcificaton associatedwith atherogenesis high expression 7 11 ANX2 role in atherogenesis 10MGP calcificaton associated with high expression 8 23 PTDGS accumulatesin end-stage 15 AGT associated with higher total atherosclerotic plaquescholesterol levels 9 10 VWF Increased levels found in 8 PECAM-implicated in migration of atherosclerosis cells under O2 stress 10 15PLIN functions in lipid metabolism 9 MSR1 deposits cholesterol duringatherogenesis 11 7 PON3 associated with coronary heart 6 CEL implicatedin the progression disease and cholesterol levels of atherosclerosis 1211 COL6A3 promoting platelet aggregation 11 MGP calcificaton associatedwith high expression 13 9 COL5A2 promoting platelet aggregation 8 COL6A2promoting platelet aggregation 14 5 APOA/B accumulation of plasma LDL in5 AT3 deficiency causes recurrent atherogenesis venous thrombosis 15 27SM22 downregulated during 25 COL6A2 promoting platelet aggregationatherogenesis 16 34 COL3A1 promoting platelet aggregation 22 SPARCcalcification of plaques 17 9 MGP calcificaton associated with 9 CD36receptor for oxidised LDL high expression 18 11 SM22 downregulatedduring 10 A2M retains LDL in core of plaque atherogenesis 19 11 SAPassociated with amyloid P in 9 APOC4 alters lipid metabolism towardlesions hypertriglyceridaemia 20 19 PLIN functions in lipid metabolism14 CD36 receptor for oxidised LDL 21 11 CD36 receptor for oxidised LDL 8PLIN functions in lipid metabolism 22 9 AT3 deficiency causes recurrent8 APOA2 associated with increased venous thrombosis atheroscleroticlesions 23 8 APOC3 associated with plasma 7 SAP associated with amyloidP in triglyceride and hyperlipidemia lesions 24 10 APOA/B accumulationof plasma LDL in 8 FGB fibrin deposition in plaque atherogenesis 25 48CNN1 modulation of SMC, a feature 26 COL6A2 promoting plateletaggregation of atherosclerosis

[0219] TABLE 6 Cardiovascular transcript images for the coexpressedcDNAs SEQ ID Library cDNAs Description of Sample Abundance % Abundance 3SMCRUNT01 3472 renal vein, smooth muscle cells, 57M, Untx 1 0.0288ENDVUNT01 5215 microvascular, dermal, endothelial cells, 22F, Untx 10.0192 HEAANOT01 12578 heart, coronary artery, CAD, 46M 1 0.0080 4MONOTXN05 2709 periph blood, monocytes, 42F, t/IL-10, LPS, NORM 1 0.0369MONOTXT02 3554 periph blood, monocytes, 42F, t/IL-10, LPS 1 0.0281MCLRUNT01 6149 periph blood mononuclear cells, 60M, untreated 1 0.0163 5ARTANOT06 6311 aorta, adventitia, 48M 2 0.0317 HEAONOT02 3482 heart,aorta, 10M 1 0.0287 HEAONOT04 4002 heart, aorta, 12F 1 0.0250 ARTANOT075716 aorta, adventitia, 65F 1 0.0175 6 HEAONOE01 3639 heart, aorta, 39M,5RP 10 0.2748 HEAONOT03 3720 heart, aorta, aw/cerebral agenesis, 27F 30.0806 HEAPNOT01 3502 heart, coronary artery, plaque, pool 2 0.0571 8ENDVTXT01 1882 microvascular, dermal, endothelial cells, 22F, t/bFGF, EF3 0.1594 ENDVTXT02 1876 microvascular, dermal, endothelial cells, 22F,t/VEGE, EF 1 0.0533 ENDVUNT01 5215 microvascular, dermal, endothelialcells, 22F, Untx 1 0.0192 13 SMCCNOS01 3494 coronary artery, smoothmuscle cells, 3M, t/TNF, IL-1, SUB 1 0.0286 SMCANOT01 7327 aortic smoothmuscle line, M 1 0.0136 15 SMCRTXT01 3453 renal vein, smooth musclecells, 57M t/TNF, IL1 2 0.0579 SMCRUNT01 3472 renal vein, smooth musclecells, 57M, Untx 1 0.0288 16 SMCCNOT01 4266 coronary artery, smoothmuscle cells, 3M 5 0.1172 SMCCNOT02 3980 coronary artery, smooth musclecells, 3M, t/TNF, IL-1 2 0.0503 SMCCNOS01 3494 coronary artery, smoothmuscle cells, 3M, t/TNF, IL-1, SUB 1 0.0286 17 ENDIUNT01 3582 iliacartery, endothelial cells, F, control, untreated 26 0.7259 ENDITXT013464 iliac artery, endothelial cells, F, t/1% oxygen 24 hr 21 0.6062ENDINOT02 3208 iliac artery, endothelial cells, F, t/TNF, IL-1 20 hr 100.3117 18 ENDVTXT01 1882 microvascular, dermal, endothelial cells, 22F,t/bFGF, EF 1 0.0531 ENDVNOT01 4955 microvascular, dermal, endothelialcells, 18F, untreated 1 0.0202 19 ENDATXP01 1877 aorta, endothelialcells, t/TNF, TIGR 1 0.0533 ENDVTXT02 1876 microvascular, dermal,endothelial cells, 22F, t/VEGF, EF 1 0.0533 ENDVTXT01 1882microvascular, dermal, endothelial cells, 22F, t/bFGF, EF 1 0.0531ENDVNOT01 4955 microvascular, dermal, endothelial cells, 18F, untreated1 0.0202 20 HEAANOT01 12578 heart, coronary artery, CAD, 46M 13 0.1034HEAONOT05 3959 heart, aorta, 17F 3 0.0758 HEAONOT04 4002 heart, aorta,12F 1 0.0250 22 ENDITXT01 3464 iliac artery, endothelial cells, F, t/1%oxygen 24 hr 1 0.0289 HEAONOE01 3639 heart, aorta, 39M, 5RP 1 0.0275HEAANOT01 12578 heart, coronary artery, CAD, 46M 1 0.0080

[0220] TABLE 7 Microarray data from cardiovascular experiments SEQ IDGEM Log2 (Cy5/Cy3) Cy3 Sample Cy5 Sample 1 LG1 −1.01 ECV304 Line, UntxECV304 Line, t/PMA + Iono 2 HG1 −1.03 HUVEC Cells, Untx, HUVEC Cells,t/TNFa 10 ng/mL Nrml 1 hr, Nrml 2 HG1 −1.03 HUAEC Cells, Untx, HUAECCells, t/TNFa 10 ng/mL Nrml 24 hr, Nrml 2 HG1 −1.03 HUVEC Cells, Untx,HUVEC Cells, t/IL1b + TNFa Nrml 10 ng/mL, 10 ng/mL 2 4hr, 4 hr, Nrml 2HG1 −1.07 HUVEC Cells, Untx, HUVEC Cells, t/PMA + TNFa Nrml, 24 hr 10nM, 10 ng/mL 24 hr, 1 hr, Nrml 2 HG1 −1.07 HUVEC Cells, Untx, HUVECCells, t/TNFa 10 ng/mL Nrml 48 hr, Nrml 2 HG1 −1.10 HUVEC Cells, UntxHUVEC Cells, t/IL4 10 ng/mL 24 hr 2 HG1 −1.10 HUVEC Cells, Untx, HUVECCells, t/TNFa 10 ng/mL Nrml 2 hr, Nrml 2 HG1 −1.12 HUVEC Cells, Untx,HUVEC Cells, t/IL4 + TNFa 48 hr 10 ng/mL, 10 ng/mL 24 hr, 1 hr 2 HG1−1.12 HUVEC Cells, Untx HUVEC Cells, t/IL10 + TNFa 10 ng/mL, 10 ng/mL 24hr,4 hr 2 HG1 −1.13 HUAEC Cells, Untx, HUAEC Cells, t/TNFa 10 ng/mL Nrml4 hr, Nrml 2 HG1 −1.14 HUVEC Cells, Untx, HUVEC Cells, t/CHX + TNFa Nrml10 mcg/mL, 10 ng/mL 30 min, 24 hr, Nrml 2 HG1 −1.14 HUVEC Cells, Untx,HUVEC Cells, t/PMA 10 nM Nrml, 24 hr 24 hr, Nrml 2 HG1 −1.15 HUVECCells, Untx, HUVEC Cells, t/TNFa 10 ng/mL Nrml 24 hr, Nrml 2 HG1 −1.17HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL Nrml 24 hr, Nrml 2 HG1−1.18 HUVEC Cells, Untx, HUVEC Cells, t/8ClcAMP + TNFa Nrml, 24 hr 7.5microM, 10 ng/mL 24 hr, 4 hr, Nrml 2 HG1 −1.19 HUVEC Cells, Untx, HUVECCells, t/TNFa 10 ng/mL Nrml 24 hr, Nrml 2 HG1 −1.21 HUVEC Cells, Untx,HUVEC Cells, t/TL1b + TNFa Nrml 10 ng/mL, 10 ng/mL 2 4hr, 24 hr, Nrml 2HG1 −1.22 HIAEC Cells, Untx, HIAEC Cells, t/TNFa 10 ng/mL Nrml 24 hr,Nrml 2 HG1 −1.22 HUVEC Cells, Untx, HUVEC Cells, t/TNFa + TNFa Nrml, 24hr .1 ng/ml, 10 ng/ml 24 hr, 24 hr, Nrml 2 HG1 −1.23 HUVEC Cells, UntxHUVEC Cells, t/TNFa 10 ng/mL 4 hr” 2 HG1 −1.26 HUVEC Cells, Untx, HUVECCells, t/8ClcAMP + TNFa Nrml, 24 hr 7.5 microM, 10 ng/mL 24 hr, 24 hr,Nrml 2 HG1 −1.28 HUVEC Cells, Untx HUVEC Cells, t/TNFa .1 ng/mL 4 hr 2HG1 −1.29 HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL Nrml 4 hr,Nrml 2 HG1 −1.29 HIAEC Cells, Untx, HIAEC Cells, t/TNFa 10 ng/mL Nrml 4hr, Nrml 2 HG1 −1.30 HUVEC Cells, Untx HUVEC Cells, t/IL10 + TNFa 10ng/mL, 10 ng/mL 24 hr, 24 hr 2 HG1 −1.31 HAEC Cells, Untx, HAEC Cells,t/TNFa 10 ng/ml Nrml 10 hr, Nrml 2 HG1 −1.31 HAEC Cells, Untx, HAECCells, t/TNFa 10 ng/ml Nrml 24 hr, Nrml 2 HG1 −1.34 HUVEC Cells, Untx,HUVEC Cells, t/lL4 + TNFa Nrml, 24 hr 10 ng/ml, 10 ng/ml 24 hr, 4 hr,Nrml 2 HG1 −1.36 HAEC Cells, Untx, HAEC Cells, t/TNFa 10 ng/ml Nrml 4hr, Nrml 2 HG1 −1.40 HUVEC Cells, Untx, HUVEC Cells, t/IL4 + TNFa Nrml,24 hr 10 ng/ml, 10 ng/ml 24 hr, 24 hr, Nrml 2 HG1 −1.43 HUVEC Cells,Untx HUVEC Cells, t/TNFa 10 ng/mL 24 hr 2 HG1 −1.44 HUVEC Cells, Untx,HUVEC Cells, t/IL1b 10 ng/mL Nrml 24 hr, Nrml 2 HG1 −1.48 HUVEC Cells,Untx HUVEC Cells, t/TNFa 10 ng/mL 24 hr” 2 HG1 −1.52 HUAEC Cells, Untx,HUAEC Cells, t/TNFa 10 ng/mL Nrml 8 hr, Nrml 2 HG1 −1.53 HUVEC Cells,Untx, HUVEC Cells, t/TNFa 10 ng/mL Nrml 4 hr, Nrml 2 HG1 −1.53 HUVECCells, Untx HUVEC Cells, t/CHX + TNFa 10 mcg/ml, 10 ng/ml 30 min, 23.5hr 2 HG1 −1.53 HUVEC Cells, Untx HUVEC Cells, t/TNFa 1 ng/mL 24 hr 2 HG1−1.55 HAEC Cells, Untx, HAEC Cells, t/TNFa 10 ng/ml Nrml 8 hr, Nrml 2HG1 −1.58 HUVEC Cells, Untx, HUVEC Cells, t/PD98059 + TNFa Nrml, 24 hr50 microM, 10 ng/mL 24 hr,24 hr, Nrml 2 HG1 −1.59 HUVEC Cells, Untx,HUVEC Cells, t/TNFa 10 ng/mL Nrml 3 d, Nrml 2 HG1 −1.59 HAEC Cells,Untx, HAEC Cells, t/TNFa 10 ng/ml Nrml 6 hr, Nrml 2 HG1 −1.61 HUVECCells, Untx, HUVEC Cells, t/IL1b + TNFa Nrml 10 ng/mL, 10 ng/mL 24 hr, 1hr, Nrml 2 HG1 −1.62 HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mlNrml, 24 hr 24 hr, Nrml 2 HG1 −1.72 HUVEC Cells, Untx, HUVEC Cells,t/TNFa 10 ng/mL Nrml 8 hr, Nrml 2 HG1 −1.80 HUVEC Cells, Untx, HUVECCells, t/PD98059 + TNFa Nrml, 24 hr 50 microM, 10 ng/mL 24 hr, 4 hr,Nrml 2 HG1 −1.83 HIAEC Cells, Untx, HIAEC Cells, t/TNFa 10 ng/mL Nrml 8hr, Nrml 2 HG1 −2.07 HUVEC Cells, Untx, HUVEC Cells, t/IL4 + TNFa 48 hr10 ng/mL, 10 ng/mL 24 hr, 24 hr 2 HG1 −2.15 HUVEC Cells, Untx, HUVECCells, t/TNFa 10 ng/mL Nrml 8 hr, Nrml 2 HG1 −2.18 HUVEC Cells, Untx,HUVEC Cells, t/IL4 + TNFa 48 hr 10 ng/mL, 10 ng/mL 24 hr,4 hr 10 UG13.38 HUVEC Cells, Untx, HUVEC Cells, t/CHX + TNFa Nrml 10 mcg/mL, 10ng/mL 30 min, 4 hr, Nrml 10 UG1 3.16 HUVEC Cells, Untx, HUVEC Cells,t/CHX + TNFa Nrml 10 mcg/mL, 10 ng/mL 30 min, 1 hr, Nrml 10 UG1 2.85HUAEC Cells, Untx, HUAEC Cells, t/TNFa 10 ng/mL Nrml 2 hr, Nrml 10 UG12.68 HPAEC Cells, Untx, HPAEC Cells, t/TNFa 10 ng/mL Nrml 1 hr, Nrml 10UG1 2.64 HPAEC Cells, Untx, HPAEC Cells, t/TNFa 10 ng/mL Nrml 2 hr, Nrml10 UG1 2.60 HUVEC Cells, Untx, HUVEC Cells, t/PMA + TNFa Nrml, 24 hr 100nM, 10 ng/mL 24 hr, 1 hr, Nrml 10 UG1 2.55 HUVEC Cells, Untx, HUVECCells, t/PMA + TNFa Nrml, 24 hr 100 nM, 10 ng/mL 24 hr, 24 hr, Nrml 10UG1 2.47 HUVEC Cells, Untx, HUVEC Cells, t/Dex + TNFa 0 hr, Nrml 100 nM,10 ng/mL 24 hr, 1 hr Nrml 10 UG1 2.40 HUVEC Cells, Untx, HUVEC Cells,t/TNFa 10 ng/mL Nrml 48 hr, Nrml 10 UG1 2.39 HUVEC Cells, Unix, HUVECCells, t/Dex + TNFa Nrml, 0 hr 100 nM, 10 ng/mL 24 hr, 24 hr Nrml 10 UG12.27 HUVEC Cells, Untx, HUVEC Cells, t/IL10 + TNFa Nrml 10 ng/mL, 10ng/mL 24 hr, 24 hr, Nrml 10 UG1 2.24 HUAEC Cells, Untx, HUAEC Cells,t/TNFa 10 ng/mL Nrml 4 hr, Nrml 10 UG1 2.18 HUAEC Cells, Untx, HUAECCells, t/TNFa 10 ng/mL Nrml 1 hr, Nrml 10 UG1 2.17 HUVEC Cells, Untx,HUVEC Cells, t/Dex + TNFa Nrml, 0 hr 10 nM, 10 ng/mL 24 hr, 24 hr Nrml10 UG1 2.14 HUVEC Cells, Untx, HUVEC Cells, t/Lipom + TNFa 24 hr 8mcg/mL, 10 ng/ml 4 hr, 4 hr, 1 hr 10 UG1 2.11 HUVEC Cells, Untx, HUVECCells, t/ILlb + TNFa Nrml 10 ng/mL, 10 ng/mL 24 hr, 24 hr, Nrml 10 UG12.04 HUVEC Cells, Untx, HUVEC Cells, t/IFNg + TNFa Nrml 200 ng/mL, 10ng/mL 24 hr, 24 hr, Nrml 10 UG1 1.95 HUVEC Cells, Untx, HUVEC Cells,t/Dex + TNFa Nrml, 0 hr 10 nM, 10 ng/mL 24 hr, 1 hr Nrml 10 UG1 1.91HUVEC Cells, Untx, HUVEC Cells, t/Lipom + 24 hr asOligo + TNFa 8 mcg/mL,100 nM, 50 nM, 10 ng/ml 4 hr, 4 hr, 1 hr 10 UG1 1.91 HUVEC Cells, Untx,HUVEC Cells, t/PD98059 + TNFa Nrml, 24 hr 50 microM, 10 ng/mL 24 hr, 1hr, Nrml 10 UG1 1.88 HUVEC Cells, Untx, HUVEC Cells, t/Dex + TNFa Nrml,0 hr 100 nM, 10 ng/mL 24 hr, 4 hr Nrml 10 UG1 1.88 HUVEC Cells, Untx,HUVEC Cells, t/Dex + TNFa Nrml, 0 hr 10 nM, 10 ng/mL 24 hr, 4 hr Nrml 10UG1 1.86 HUVEC Cells, Untx, HUVEC Cells, t/IL4 + TNFa 48 hr 10 ng/mL, 10ng/mL 24 hr, 24 hr 10 UG1 1.82 HUVEC Cells, Untx, HUVEC Cells, t/Lipom +TNFa 24 hr 8 mcg/mL, 10 ng/ml 4 hr, 4 hr, 24 hr 10 UG1 1.82 HUVEC Cells,Untx, HUVEC Cells, t/IL4 + TNFa 48 hr 10 ng/mL, 10 ng/mL 24 hr, 1 hr 10UG1 1.76 HUVEC Cells, Untx, HUVEC Cells, t/IL1b 10 ng/mL Nrml 24 hr,Nrml 10 UG1 1.74 HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL Nrml 24hr, Nrml 10 HG3 1.20 HUAEC Cells, Untx, HUAEC Cells, t/TNFa 10 ng/mLNrml 1 hr, Nrml 10 HG3 1.04 HUAEC Cells, Untx, HUAEC Cells, t/TNFa 10ng/mL Nrml 2 hr, Nrml 12 HG5 1.02 ECV304 Line, t/TNFa ECV304 Line, Untx,Nrml 10 ng/mL 3 d, Nrml 12 HG5 −1.07 HUVEC Cells, Untx HUVEC Cells,t/IL10 + TNFa 10 ng/mL, 10 ng/mL 24 hr, 4 hr 12 HG5 −1.16 HUAEC Cells,Untx, HUAEC Cells, t/TNFa 10 ng/mL Nrml 8 hr, Nrml 12 HG5 −1.18 HMVECCells, Untx, HMVEC Cells, t/TNFa 10 ng/mL Nrml 24 hr, Nrml 12 HG5 −1.24HMVEC Cells, Untx, HMVEC Cells, t/TNFa 10 ng/mL Nrml 8 hr, Nrml 12 HG5−1.50 HIAEC Cells, Untx, HIAEC Cells, t/TNFa 10 ng/mL Nrml 8 hr, Nrml 18HG2 −2.38 HUVEC Cells, Untx, HUVEC Cells, t/IFNg + TNFa Nrml 10 ng/mL,10 ng/mL 24 hr, 24 hr, Nrml 32 HG3 −1.13 HUVEC Cells, Untx HUVEC Cells,t/PMA + TNFa 24 hr, Nrml 10 nM, 10 ng/mL 24 hr, 24 hr, Nrml 33 LG1 −1.17Tangier Fibroblast Cells, t/LDL Cholesterol, Nrml 34 UG1 −1.04 ECV304Line, Untx ECV304 Line, t/PMA + Iono 1 microM, 1 meg/ml 4 hr 34 UG1−1.11 ECV304 Line, Untx ECV304 Line, t/PMA + Iono 1 microM, 1 meg/ml 5hr

[0221]

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 26 <210> SEQ ID NO 1<211> LENGTH: 1334 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: misc_feature <220> FEATURE: <221> NAME/KEY:unsure <222> LOCATION: 674, 735, 788 <223> OTHER INFORMATION: a, t, c,g, or other <400> SEQUENCE: 1 aggcctccct ccacctgtct tctcagagcagataatggca agcatggctg ccgtgctcac 60 ctgggctctg gctcttcttt cagcgttttcggccacccag gcacggaaag gcttctggga 120 ctacttcagc cagaccagcg gggacaaaggcagggtggag cagatccatc agcagaagat 180 ggctcgcgag cccgcgaccc tgaaagacagccttgagcaa gacctcaaca atatgaacaa 240 gttcctggaa aagctgaggc ctctgagtgggagcgaggct cctcggctcc cacaggaccc 300 ggtgggcatg cggcggcagc tgcaggaggagttggaggag gtgaaggctc gcctccagcc 360 ctacatggca gaggcgcacg agctggtgggctggaatttg gagggcttgc ggcagcaact 420 gaagccctac acgatggatc tgatggagcaggtggccctg cgcgtgcagg agctgcagga 480 gcagttgcgc gtggtggggg aagacaccaaggcccagttg ctggggggcg tggacgaggc 540 ttgggctttg ctgcagggac tgcagagccgcgtggtgcac cacaccggcc gcttcaaaga 600 gctcttccac ccatacgccg agagcctggtgagcggcatc gggcgccacg tgcaggagct 660 gcaccgcagt gtgntccgca cgcccccgccagccccgcgc gcctcagtcg ctgcgtgcag 720 gtgctctccc ggaantcacg ctcaaggccaaggccctgca cgcacgcatc cagcagaacc 780 tggaccantg cgcgaagagc tcagcagagcctttgcaggc actgggactg aggaaggggc 840 cggcccggac ccccagatgc tctccgaggaggtgcgccag cgacttcagg ctttccgcca 900 ggacacctac ctgcagatag ctgccttcactcgcgccatc gaccaggaga ctgaggaggt 960 ccagcagcag ctggcgccac ctccaccaggccacagtgcc ttcgccccag agtttcaaca 1020 aacagacagt ggcaaggttc tgagcaagctgcaggcccgt ctggatgacc tgtgggaaga 1080 catcactcac agccttcatg accagggccacagccgtctg ggggacccct gaggatctac 1140 ctgcccaggc ccattcccag cttcttgtctggggagcctt ggctctgagc ctctagcatg 1200 gttcagtcct tgaaagtggc ctgttgggtggagggtggaa ggtcctgtgc aggacaggga 1260 ggccaccaaa ggggctgctg tctcctgcatatccagcctc ctgcgactcc ccaatgcagg 1320 atgcattcat tcac 1334 <210> SEQ IDNO 2 <211> LENGTH: 1702 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<220> FEATURE: <221> NAME/KEY: misc_feature <400> SEQUENCE: 2 cgttcccactgcaccctgga gaacgagcct ttgcggggtt tctcctggct gtcctccgac 60 cccggcggtctcgaaagcga cacgctgcag tgggtggagg agccccaacg ctcctgcacc 120 gcgcggagatgcgcggtact ccaggccacc ggtggggtcg agcccgcagg ctggaaggag 180 atgcgatgccacctgcgcgc caacggctac ctgtgcaagt accagtttga ggtcttgtgt 240 cctgcgccgcgccccggggc cgcctctaac ttgagctatc gcgcgccctt ccagctgcac 300 agcgccgctctggacttcag tccacctggg accgaggtga gtgcgctctg ccggggacag 360 ctcccgatctcagttacttg catcgcggac gaaatcggcg ctcgctggga caaactctcg 420 ggcgatgtgttgtgtccctg ccccgggagg tacctccgtg ctggcaaatg cgcagagctc 480 cctaactgcctagacgactt gggaggcttt gcctgcgaat gtgctacggg cttcgagctg 540 gggaaggacggccgctcttg tgtgaccagt ggggaaggac agccgaccct tggggggacc 600 ggggtgcccaccaggcgccc gccggccact gcaaccagcc ccgtgccgca gagaacatgg 660 ccaatcagggtcgacgagaa gctgggagag acaccacttg tccctgaaca agacaattca 720 gtaacatctattcctgagat tcctcgatgg ggatcacaga gcacgatgtc tacccttcaa 780 atgtcccttcaagccgagtc aaaggccact atcaccccat cagggagcgt gatttccaag 840 tttaattctacgacttcctc tgccactcct caggctttcg actcctcctc tgccgtggtc 900 ttcatatttgtgagcacagc agtagtagtg ttggtgatct tgaccatgac agtactgggg 960 cttgtcaagctctgctttca cgaaagcccc tcttcccagc caaggaagga gtctatgggc 1020 ccgccgggcctggagagtga tcctgagccc gctgctttgg gctccagttc tgcacattgc 1080 acaaacaatggggtgaaagt cggggactgt gatctgcggg acagagcaga gggtgccttg 1140 ctggcggagtcccctcttgg ctctagtgat gcatagggaa acaggggaca tgggcactcc 1200 tgtgaacagtttttcacttt tgatgaaacg gggaaccaag aggaacttac ttgtgtaact 1260 gacaatttctgcagaaatcc cccttcctct aaattccctt tactccactg aggagctaaa 1320 tcagaactgcacactccttc cctgatgata gaggaagtgg aagtgccttt aggatggtga 1380 tactgggggaccgggtagtg ctggggagag atattttctt atgtttattc ggagaatttg 1440 gagaagtgattgaacttttc aagacattgg aaacaaatag aacacaatat aatttacatt 1500 aaaaaataatttctaccaaa atggaaagga aatgttctat gttgttcagg ctaggagtat 1560 attggttcgaaatcccaggg aaaaaaataa aaataaaaaa ttaaaggatt gttgataaaa 1620 aaaaaaaaaaaaaaagatct ttaattaagc ggcccaagct tattcccttt agtgagggtt 1680 aattttagcttgcactggcc ac 1702 <210> SEQ ID NO 3 <211> LENGTH: 586 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature<220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: 48, 66, 560, 574,577, 580 <223> OTHER INFORMATION: a, t, c, g, or other <400> SEQUENCE: 3tcgaggactc cgccaactac agctgcgtct acgtggacct gaagccgnct ttcgggggct 60acgcgnccag cgagcgcttg gagctgcacg tggacggacc ccctcccagg cctcagctcc 120gggcgacgtg gagtggggcg gtcctggcgg gccgagatgc cgtcctgcgc tgcgagggac 180ccatccccga cgtcaccttc gagctgctgc gcgagggcga gacgaaggcc gtgaagacgg 240tccgcacccc cggggccgcg gcgaacctcg agctgatctt cgtggggccc cagcacgccg 300gcaactacag gtgccgctac cgctcctggg tgccccacac cttcgaatcg gagctcagcg 360accctgtgga gctcctggtg gcagaaagct gatgcagccg cgggcccagg gtgctgttgg 420tgtcctcaga agtgccgggg attctggact ggctccctcc cctcctgttg cagcacaagg 480ccggggtctc tggggggctg gagaagcctc cctcattcct cccaggaatt aataaatgtg 540aagagagctc tgtttaaaan aaaaaaaaag aaanaanaan aaccaa 586 <210> SEQ ID NO 4<211> LENGTH: 433 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: misc_feature <400> SEQUENCE: 4 ctcaagacccagcagtggga cagccagaca gacggcacga tggcactgag ctcccagatc 60 tgggccgcttgcctcctgct cctcctcctc ctcgccagcc tgaccagtgg ctctgttttc 120 ccacaacagacgggacaact tgcagagctg caaccccagg acagagctgg agccagggcc 180 agctggatgcccatgttcca gaggcgaagg aggcgagaca cccacttccc catctgcatt 240 ttctgctgcggctgctgtca tcgatcaaag tgtgggatgt gctgcaagac gtagaaccta 300 cctgccctgcccccgtcccc tcccttcctt atttattcct gctgccccag aacataggtc 360 ttggaataaaatggctggtt cttttgtttt ccaaaaaaaa aaaaaaaaaa aaaaaaaaaa 420 aaaaaaaaaaaaa 433 <210> SEQ ID NO 5 <211> LENGTH: 3111 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <220>FEATURE: <221> NAME/KEY: unsure <222> LOCATION: 44 <223> OTHERINFORMATION: a, t, c, g, or other <400> SEQUENCE: 5 cgagggcggacgcaaagaac gcggaggacc tctgggtgcc tgcnggggag ctgctccagc 60 cgggccgccgggagcggtgg ggagagcatc gcgcagccgc ccctccacgc gcccgcccag 120 ccgcgttcgcccactgggct ctcccggctg cagtgccagg gcgcaggacg cggccgatct 180 cccgctcccgccacctccgc caccatgctg ctcccccagc tctgctggct gccgctgctc 240 gctgggctgctcccgccggt gcccgctcag aagttctcgg cgctcacgtt tttgagagtg 300 gatcaagataaagacaagga ttgtagcttg gactgtgcgg gttcgcccca gaaacctctc 360 tgcgcatctgacggaaggac cttcctttcc cgttgtgaat ttcaacgtgc caagtgcaaa 420 gatccccagctagagattgc atatcgagga aactgcaaag acgtgtccag gtgtgtggcc 480 gaaaggaagtatacccagga gcaagcccgg aaggagtttc agcaagtgtt cattcctgag 540 tgcaatgacgacggcaccta cagtcaggtc cagtgtcaca gctacacggg atactgctgg 600 tgcgtcacgcccaacgggag gcccatcagc ggcactgccg tggcccacaa gacgccccgg 660 tgcccgggttccgtaaatga aaagttaccc caacgcgaag gcacaggaaa aacagatgat 720 gccgcagctccagcgttgga gactcagcct caaggagatg aagaagatat tgcatcacgt 780 taccctaccctttggactga acaggttaaa agtcggcaga acaaaaccaa taagaattca 840 gtgtcatcctgtgaccaaga gcaccagtct gccctggagg aagccaagca gcccaagaac 900 gacaatgtggtgatccctga gtgtgcgcac ggcggcctct acaagccagt gcagtgccac 960 ccctccacggggtactgctg gtgcgtcctg gtggacacgg ggcgccccat tcccggcaca 1020 tccacaaggtacgagcagcc gaaatgtgac aacacgggcc agggcccacc cagccaaagc 1080 ccgggacctgtacaagggcc gccagctaca aggttgtccg ggtgccaaaa agcatgagtt 1140 tctgaccagcgttctggacg cgctgtccac ggacatggtc cacgccgcct ccgacccctc 1200 ctcctcgtcaggcaggctct cagaacccga ccccagccat accctagagg agcgggtggt 1260 gcactggtacttcaaactac tggataaaaa ctccagtgga gacatcggca aaaaggaaat 1320 caaacccttcaagaggttcc ttcgcaaaaa atcaaagccc aaaaaatgtg tgaagaagtt 1380 tgttgaatactgtgacgtga ataatgacaa atccatctcc gtacaagaac tgatgggctg 1440 cctgggcgtggcgaaagagg acggcaaagc ggacaccaag aaacgccaca cccccagagg 1500 tcatgctgaaagtacgtcta atagacagcc aaggaaacaa ggataaatgg ctcatacccc 1560 gaaggcagttcctagacaca tgggaaattt ccctcaccaa agagcaatta agaaaacaaa 1620 aacagaaacacatagtattt gcactttgta ctttaaatgt aaattcactt tgtagaaatg 1680 agctatttaaacagactgtt ttaatctgtg aaaatggaga gctggcttca gaaaattaat 1740 cacataccaatgtatgtgtc ctcttttgac cttggaaatc tgtatgtggt ggagaagtat 1800 ttgaatgcatttaggcttaa tttcttcgcc ttccacatgt taacagtaga gctctatgca 1860 ctccggctgcaatcgtatgg ctttctctaa cccctgcagt cacttccaga tgcctgtgct 1920 tacagcattgtggaatcatg ttggaagctc cacatgtcca tggaagtttg tgatgtacgg 1980 ccgaccctacaggcagttaa catgcatggg ctggtttgtt tcttgggatt ttctgttagt 2040 ttgtcttgttttgctttcca gagatcttgc tcatacaatg aatcacgcaa ccactaaagc 2100 tatccagttaagtgcaggta gttcccctgg aggaaataat attttcaaac tgtcgttggt 2160 gtgatactttggctcaaagg atctttgctt ttccatttta agcttctgtt ttgagttttg 2220 ccctggggcttgaatgagtc ccagagagtc gttcggatgg tgggaggctg cctaggaggc 2280 agtaaatccagttcacagtg cctgggaggg gcccatcctt ccaaaatgta aatccagttc 2340 gcggtgtgaccgagctgggc taacaggctt gtctgcctgg ttttcctacc tacacgtgga 2400 cattattctcctgatcctcc tacctggttc caccccaggg ctaccggaag gtaaaatctt 2460 cacctgaaccaattatgagc agtctcctta ctgaaggtac agccggatac gtggtgcccc 2520 cggggctggtgttggcagcc ggggggaggt gcctgagggt ccccacggtt cctttctgct 2580 tttctgaatgcatcaagggt acgagaactt gccaatggga aattcatccg agtggcactg 2640 gcagagaaggataggagtgg aatgcccaca cagtgaccaa cagaactggt ctgcgtgcat 2700 aaccagctgccaccctcagg cctgggcccc agagctcagg gcacccagtg tcttaaggaa 2760 ccatttggaggacagtctga gagcaggaac ttcaagctgt gattctatct cggctcagac 2820 ttttggttggaaaaagatct tcatggcccc aaatcccctg agacatgcct tgtagaatga 2880 ttttgtgatgttgtgatgct tgtggagcat cgcgtaaggc ttcttgctta tttaaactgt 2940 gcaaggtaaaaatcaagcct ttggagccac agaaccagct caagtacatg ccaatgttgt 3000 ttaagaaacagttatgatcc taaacttttt ggataatctt ttatatttct gacctttgaa 3060 tttaatcattgttcttagat taaaataaaa tatgctattg aaactaaaaa a 3111 <210> SEQ ID NO 6<211> LENGTH: 2311 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: misc_feature <220> FEATURE: <221> NAME/KEY:unsure <222> LOCATION: 474-544, 2288, 2295-2299 <223> OTHER INFORMATION:a, t, c, g, or other <400> SEQUENCE: 6 gccgctcgcc cacggactcc gacgtgtccctcgactccga ggactccggg gctaagtctc 60 caggcatcct gggctacaat atctgtccccgcgggtggaa tggcagcctt cggctcaagc 120 gtggcagcct ccccgccgag gcctcctgcaccacctagag ccccaccccc gaccccaccc 180 cgggagggca gagccagaag aaggctcattagacctgggg gacccaaagg gtctggcctc 240 tttgggcagc cccagagatg aggggtcagcagaggagagc tctggggttg gggatgggtt 300 agggacgcaa gcttgagttc tagcccttgctctcattcag ctgttgtgtg accctgggta 360 agacccttcc ttgtttgacc ctcagctttcccatctgttt aatggtggct ttggccaagg 420 caatccacaa acgtcaaaat tccccttcccatcagtacac acaccgatgc acannnnnnn 480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 540 nnnntagtta gtgccttgga tgaggcggggcagtgtgtat atggacccct ggacttgcta 600 ccttcagggt tccatactcg tccctcccctcctggctctg ctgtctggag tctggcaagc 660 ggggtgtgtt cagaaggtcc taggcctgtgtcgcatgtcc aggcactggc ctgaccatcc 720 ggctccctgg gcaccaagtc ccagggcaggagcagctgtt ttccatccct tcccagacaa 780 gctctatttt tatcacaatg acctttagagaggtctccca ggccagctca aggtgtccca 840 ctatcccctc tggagggaag aggcaggaaaattctccccg ggtccctgtc atgctacttt 900 ctccatccca gttcagactg tccaggacatcttatctgca gccataagag aattataagg 960 cagtgatttc ccttaggccc aggacttgggcctccagctc atctgttcct tctgggccca 1020 ttcatgggca ggttctgggc tcaaagctgaactggggaga gaagagatac agagctacca 1080 tgtgacttta cctgattgcc ctcagtttggggttgcttat tgggaaagag agagacaaag 1140 agttacttgt tacgggaaat atgaaaagcatggccaggat gcatagagga gattctagca 1200 ggggacagga ttggctcaga tgacccctgagggctcttcc agtcttgaaa tgcattccat 1260 gatattagga agtcgggggt gggtggtggtggtgggctag ttgggcttga atttaggggc 1320 cgatgagctt gggtacgtga gcagggtgttaagttagggt ctgcctgtat ttctggtccc 1380 cttgggaaat gtccccttct tcagtgtcagacctcagtcc cagtgtccat atcgtgccca 1440 gaaaagtaga cattatcctg ccccatcccttccccagtgc actctgacct agctagtgcc 1500 tggtgcccag tgacctgggg gagcctggctgcaggccctc actggttccc taaaccttgg 1560 tggctgtgat tcaggtcccc aggggggactcagggaggaa tatggctgag ttctgtagtt 1620 tccagagttg ggctggtaga gctttctagaggttcagaat attagcttca ggatcagctg 1680 ggggtatgga attggctgag gatcaaacgtatgtaggtga aaggatacca ggatgttgct 1740 aaaggtgagg gacagtttgg gtttgggacttaccggggtg atgttagatc tggaaccccc 1800 aagtgaggct ggagggagtt aaggtcagtatggaagatag ggttgggaca gggtgctttg 1860 gaatgaaaga gtgaccttag agggctccttgggcctcagg aatgctcctg ctgctgtgaa 1920 gatgagaagg tgctcttact cagttaatgatgagtgacta tatttaccaa agcccctacc 1980 tgctgctggg tcccttgtag cacaggagactggggctaag ggcccctccc agggaaggga 2040 caccatcagg cctctggctg aggcagtagcatagaggatc catttctacc tgcatttccc 2100 agaggactag caggaggcag ccttgagaaaccggcagttc ccaagccagc gcctggctgt 2160 tctctcattg tcactgccct ctccccaacctctcctctaa cccactagag attgcctgtg 2220 tcctgcctct tgcctcttgt agaatgcagctctggccctc aataaatgct tcctgcattc 2280 taaaaaanaa aaaannnnna aaaaaaaaag g2311 <210> SEQ ID NO 7 <211> LENGTH: 1866 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <400>SEQUENCE: 7 agcttttgtt cacactttaa atagcagtcc cagaatgatt tcactacagactctctggaa 60 agcctgggag ctgaattccg gaagatcccc acatcgatga aagcaaagcgaagccaccaa 120 gccatcatca tgtccacgtc gctacgagtc agcccatcca tccatggctaccacttcgac 180 acagcctctc gtaagaaagc cgtgggcaac atctttgaaa acacagaccaagaatcacta 240 gaaaggctct tcagaaactc tggagacaag aaagcagagg agagagccaagatcattttt 300 gccatagatc aagatgtgga ggagaaaacg cgtgccctga tggccttgaagaagaggaca 360 aaagacaagc ttttccagtt tctgaaactg cggaaatatt ccatcaaagttcactgaaga 420 gaagaggatg gataaggacg ttatccaaga atggacattc aaagaccaagtgagtttgtg 480 agattctaac agatgcagca ttttgctgct accttacaag cttctcttctgtcaggactc 540 cagaggctgg aaagggaccg ggactggaaa gggaccagga ctgaacagactggttacaaa 600 gactccaaac aatttcatgc cctgtgctgt tacagaggag aacaaaatgctttcagcaag 660 gatttgaaaa ctcttccgtc cctgcaggaa aggattgatg ctgatagaagagcctggaca 720 gatgtaatga gaactaaaga aaacagatgg ctggagatga catttatccagggtcacttt 780 gtcaggccct aggacttaaa tcgaagttga actttttttt ttttttaaccaaatagatag 840 gggaagggag gagggagagg gaggacaggg agagaaaata ccatgcataaattgtttact 900 gaatttttat atctgagtgt tcaaaatatt tccaagcctg agtattgtctattggtatag 960 atttttagaa atcaataatt gattatttat ttgcacttat tacaatgcctgaaaaagtgc 1020 accacatgga tgttaagtag aaattcaaga aagtaagatg tcttcagcaactcagtaaaa 1080 ccttacgcca ccttttggtt tgtaaaaggt tttttataca tttcaaacaggttgcacaaa 1140 agttaaaata atggggtctt ttataaatcc aaagtactgt gaaaacattttacatatttt 1200 ttaaatcttc tgactaatgc taaaacgtaa tctaattaaa tttcatacagttactgcagt 1260 aagcattagg aagtgaatat gatatacaaa atagtttata aagactctatagtttctata 1320 atttatttta ctggcaaatg tcatgcaaca ataataaatt attgtaaactttgtggcttt 1380 tggtctgtga tgcttggtct caaaggaaaa aataagatgg taaatgttgatatttacaaa 1440 cttttctaaa gatgtgtctc taacaataaa agttaatttt agagtagttttatattaatt 1500 accaaacttt ttcaaaacaa attcttacgt caaatatctg ggaagtttctctgtcccaat 1560 cttaaaatat aaaatataga tatagaagtt catagattga ctccttggcatttctattta 1620 tgtatccatt aaggatgagt tttaaaaggc tttctcttca tacttttgaaaaatttcttc 1680 tatgattaca gtagctatgt acatgtgtac atctattttt cccaagcaatatgttttggg 1740 tttagagtct gagtgatgac caagattctg tgtgttacta ctgtttgtttaataggaaca 1800 aatatagaaa taatattatc tctttgctta tttcccgtta aaactataataaaatgtttc 1860 taggaa 1866 <210> SEQ ID NO 8 <211> LENGTH: 1929 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:misc_feature <400> SEQUENCE: 8 gctgcctgcc ggtgctcttc gtggctctgggcatggcctc ggaccccatc ttcacgctgg 60 cgcccccgct gcattgccac tacggggccttcccccctaa tgcctctggc tgggagcagc 120 ctcccaatgc cagcggcgtc agcgtcgccagcgctgccct agcagccagc gccgccagcc 180 gtgtcgccac cagtaccgac ccctcgtgcagcggcttcgc cccgccggac ttcaaccatt 240 gccctcaagg attgggacta taatggccttcctgtgctca ccaccaacgc catcggccag 300 tgggatctgg tgtgtgacct gggctggcaggtgatcctgg agcagatcct cttcatcttg 360 ggctttgcct ccggctacct gttcctgggttaccccgcag acagatttgg ccgtcgcggg 420 attgtgctgc tgaccttggg gctggtgggcccctgtggag taggaggggc tgctgcaggc 480 tcctccacag gcgtcatggc cctccgattcctcttgggct ttctgcttgc cggtgttgac 540 ctgggtgtct acctgatgcg cctggagctgtgcgacccaa cccagaggct tcgggtggcc 600 ctggcagggg agttggtggg ggtgggagggcacttcctgt tcctgggcct ggcccttgtc 660 tctaaggatt ggcgattcct acagcgaatgatcaccgctc cctgcatcct cttcctgttt 720 tatggctggc ctggtttgtt cctggagtccgcacggtggc tgatagtgaa gcggcagatt 780 gaggaggctc agtctgtgct gaggatcctggctgagcgaa accggcccca tgggcagatg 840 ctgggggagg aggcccagga ggccctgcaggacctggaga atacctgccc tctccctgca 900 acatcctcct tttcctttgc ttccctcctcaactaccgca acatctggaa aaatctgctt 960 atcctgggct tcaccaactt cattgcccatgccattcgcc actgctacca gcctgtggga 1020 ggaggaggga gcccatcgga cttctacctgtgctctctgc tggccagcgg caccgcagcc 1080 ctggcctgtg tcttcctggg ggtcaccgtggaccgatttg gccgccgggg catccttctt 1140 ctctccatga cccttaccgg cattgcttccctggtcctgc tgggcctgtg ggattatctg 1200 aacgaggctg ccatcaccac tttctctgtccttgggctct tctcctccca agctgccgcc 1260 atcctcagca ccctccttgc tgctgaggtcatccccacca ctgtccgggg ccgtggcctg 1320 ggcctgatca tggctctagg ggcgcttggaggactgagcg gcccggccca gcgcctccac 1380 atgggccatg gagccttcct gcagcacgtggtgctggcgg cctgcgccct cctctgcatt 1440 ctcagcatta tgctgctgcc ggagaccaagcgcaagctcc tgcccgaggt gctccgggac 1500 ggggagctgt gtcgccggcc ttccctgctgcggcagccac cccctacccg ctgtgaccac 1560 gtcccgctgc ttgccacccc caaccctgccctctgagcgg cctctgagta ccctggcggg 1620 aggctggccc acacagaaag gtggcaagaagatcgggaag actgagtagg gaaggcaggg 1680 ctgcccagaa gtctcagagg cacctcacgccagccatcgc ggagagctca gagggccgtc 1740 cccaccctgc ctcctccctg ctgctttgcattcacttcct tggccagagt caggggacag 1800 ggagagagct ccacactgta accactgggtctgggctcca tcctgcgccc aaagacatcc 1860 acccagacct cattatttct tgctctatcattctgtttca ataaagacat ttggaataaa 1920 aaaaaaaaa 1929 <210> SEQ ID NO 9<211> LENGTH: 1831 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: misc_feature <400> SEQUENCE: 9 ctggagccgccctgggtgtc agcggctcgg ctcccgcgca cgctccggcc gtcgcgcacc 60 tcgggcacctgcaggtccgt ggcgtcccgc ggctgggcgc ccctgactcc gtcccggcca 120 gggagggccatgatttccct cccggggccc ctggtgacca acttgctgcg gtttttgttc 180 ctggggctgagtgccctcgc gcccccctcg cgggcccagc tgcaactgca cttgcccgcc 240 aaccggttgcaggcggtgga gggaggggaa gtggtgcttc cagcgtggta caccttgcac 300 ggggaggtgtcttcatccca gccatgggag gtgccctttg tgatgtggtt cttcaaacag 360 aaagaaaaggaggatcaggt gttgtcctac atcaatgggg tcacaacaag caaacctgga 420 gtatccttggtctactccat gccctcccgg aacctgtccc tgcggctgga gggtctccag 480 gagaaagactctggccccta cagctgctcc gtgaatgtgc aagacaaaca aggcaaatct 540 aggggccacagcatcaaaac cttagaactc aatgtactgg ttcctccagc tcctccatcc 600 tgccgtctccagggtgtgcc ccatgtgggg gcaaacgtga ccctgagctg ccagtctcca 660 aggagtaagcccgctgtcca ataccagtgg gatcggcagc ttccatcctt ccagactttc 720 tttgcaccagcattagatgt catccgtggg tctttaagcc tcaccaacct ttcgtcttcc 780 atggctggagtctatgtctg caaggcccac aatgaggtgg gcactgccca atgtaatgtg 840 acgctggaagtgagcacagg tcagtgaggg ggcctggagc tgcagtggtt gctggagctg 900 ttgtgggtaccctggttgga ctggggttgc tggctgggct ggtcctcttg taccaccgcc 960 ggggcaaggccctggaggag ccagccaatg atatcaagga ggatgccatt gctccccgga 1020 ccctgccctggcccaagagc tcagacacaa tctccaagaa tgggaccctt tcctctgtca 1080 cctccgcacgagccctccgg ccaccccatg gccctcccag gcctggtgca ttgaccccca 1140 cgcccagtctctccagccag gccctgccct caccaagact gcccacgaca gatggggccc 1200 accctcaaccaatatccccc atccctggtg gggtttcttc ctctggcttg agccgcatgg 1260 gtgctgtgcctgtgatggtg cctgcccaga gtcaagctgg ctctctggta tgatgacccc 1320 accactcattggctaaagga tttggggtct ctccttccta taagggtcac ctctagcaca 1380 gaggcctgagtcatgggaaa gagtcacact cctgaccctt agtactctgc ccccacctct 1440 ctttactgtgggaaaaccat ctcagtaaga cctaagtgtc caggagacag aaggagaaga 1500 ggaagtggatctggaattgg gaggagcctc cacccacccc tgactcctcc ttatgaagcc 1560 agctgctgaaattagctact caccaagagt gaggggcaga gacttccagt cactgagtct 1620 cccaggcccccttgatctgt accccacccc tatctaacac cacccttggc tcccactcca 1680 gctccctgtattgatataac ctgtcaggct ggcttggtta ggttttactg gggcagagga 1740 tagggaatctcttattaaaa ctaacatgaa atatgtgttg ttttcatttg caaatttaaa 1800 taaagatacataatgtttgt atgagataag a 1831 <210> SEQ ID NO 10 <211> LENGTH: 1453 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:misc_feature <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION:903-935 <223> OTHER INFORMATION: a, t, c, g, or other <400> SEQUENCE: 10ggagccaagt ggggccctcg gcctcttcct tcgttccagg cccatgattt tccctacact 60tctccctggc ccaggctcca gccacaggca cctctcctgc ccccgcccac cctcctgacc 120gcagctccca ggccctggag acctccaggc tttcctgccc tgggcagccc cacctcacag 180ccagagtcaa tgccttcatg ggaagggctc ccagccacac ccagagtggc ccaaagctgt 240tgaagtcagc atcctttgtc ccatcaggac cctcctgcct cctctccagg cccttgttcg 300cctccccacc ctcctcagag gcccggggaa gggaagagca ggtcagtaca gaggttctgt 360ctacagggag gggccctggg tctatgcaca gctggagctc tgagccttcc acagcccgtg 420tgactgctag agggcagggg tgcagggctc aggggggccg ggctggtcct ttggggctgg 480tgttcctacg tcagtcccca cctggggaat aaactccagc ctctcctgct catacagaag 540gaactggttg ggtttgcttt atgggatctt tgagaccaaa acagatgctc ctgtttgctg 600ggggagggtg tgagcacgga gtatttctgt ccctcgtgaa gtcacgtcac acaggggaga 660ggcgaggtcg atggaactgg ccacgcacag gctctggctc tggaaggagg gatgatgagt 720gggcgttttc ccggcaggcc cccggggtcc tcagcctcag caacccaggg agaggacaga 780aatgaaccga tggttgaggg attgtcacgg gaggaacatg acacccgaag ggactctagg 840tgccctcgga gtgccacaca tgcccagacc ttctcacacc cacacaaata ggctctgccg 900tgnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnttgtt cacactcaga acccaggaca 960gccacagcca ccgcttaggg gaagccactg cagatgcccc tggaatgggc acagcacagc 1020cagggcgctc ttccaggcag gcgaggataa cttgagagtt tcctagggca ccagggacag 1080agctcagagg cccccgaggt gtgtgtagga ggcggaggcc cgcagagcac agagcaggag 1140aagggcttgg gccctggagg agaaagccat tctggacacc aggggacctg gacggagggt 1200ccccacagcc cgtgccccac gccgcctgga ggccagaggg gtcagtggcc ctgctgtccc 1260ggctccatct tggttctagc cgccacctgt atgaacacag tggcccggct taacgcacta 1320acccagcctc tccctgtgtc ccacagggag tagcaagacc caccccacac tgccttcacc 1380atctacacca gtgacgccgc tgtgtgtctt agcatggaaa taaataaacc tgaatgcaaa 1440aaaaaaaaaa agg 1453 <210> SEQ ID NO 11 <211> LENGTH: 443 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature<400> SEQUENCE: 11 gatatagaca acttccagag tcaccagtgt gcaaatggagccacctgcat tagtcatact 60 aatggctatt cttgcctctg ttttggaaat tttacaggaaaattttgcag acagagcaga 120 ttaccctcaa cagtctgtgg gaatgagaag acaaatctcacttgctacaa tggaggcaac 180 tgcacagagt tccagactga attaaaatgt atgtgccggccaggttttac tggagaatgg 240 tgtgaaaagg acattgatga gtgtgcctct gatccgtgtgtcaatggagg tctgtgccag 300 gacttactca acaaattcca gtgcctctgt gatgttgcctttgctggcga gcgctgcgag 360 gtggacttgg cagatgactt gatctccgac attttcaccactattggctc agtgactgtc 420 gccttgttac tgatcctctt gct 443 <210> SEQ ID NO12 <211> LENGTH: 1537 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: misc_feature <220> FEATURE: <221> NAME/KEY:unsure <222> LOCATION: 284-285, 287 <223> OTHER INFORMATION: a, t, c, g,or other <400> SEQUENCE: 12 aaaaaaaaca acccggtagc attgtccctt ccccactgacaaacttatca aatccagaag 60 ctttagagtt tcgtctctaa ttatttttct cctgaacaaaattacccaag tcaaaacaaa 120 atgtattttt agaattacgg cagcatacga cctgaattttgtgagtttcg tggctttatc 180 ttaaatcacc atttccctaa aaacggtttc tttctccttagaaatgctgg tggcaacttg 240 atgaaacagc caaatgcacc agggcaggtc actttcccaaaaannanaag aaaaaaaact 300 cattgagata gctacagttc tataggttaa tttaaagcctcctttttcta ctcatttttg 360 aaagcaaaat tacattttac tattttacat aaccagtgaaaagacgttga aagcctacag 420 ctcactgttt ttggtgctct ggaaatgttg agggtgggtttttaaccagt gatttttaac 480 gtgcagtgaa tttgttagac ttttaaacac cagctaaggtagtcaaactt gatccccatt 540 aaaaatcaag gaattagggg tcgggggagg gtttaggagtgatccagaat gacctcccag 600 aattactgtg cgtacaactt tatttttcag agttttcattggaatggtaa gagttttatg 660 aaagacagtt ttaaaactta ttctgagtta aatattaatactttaaaaaa ttattgtact 720 agacttattg cagccttttg aaagtagcag agtttcatcataccacatat ataacagagc 780 ataaattttc tataatcagg caccttttgc tgcttttgagtaagactgtt ttcctgttta 840 ggtgttaagc atcgccagac ataaaaatct attctctcctctcgattgta gcatagcctg 900 acagctctag atacagcatt tctatgatga aaaatgagtatccatcagga aatctagaag 960 actagccgtg ttttctcaga ctccaccttt gtttgcactctgttgcctgt gaggagcttt 1020 ctggcatgtg attatttact tcaaaactag agttccaagcacctacatta attattttat 1080 attgtgtgca gaatagtata tcttttaatg tcagatatgatacactgcac atattgcttt 1140 tgcactctta aaatttttgt actaaataat agaaaatatttatattcttt gagtgtgagc 1200 tttgaataga tggcattatc actttattgt ttttttaacaaaaacttttt ctcaattatt 1260 ctattgcaat gttattctga gcaagtccta tgccaaatatcttgtataat gtttgtatgg 1320 aagattaaat tttactcttg tgtggtaaga ctatttcagttactgatttt atagttggaa 1380 tttgatattc cagcacaaag tccacagtgt attcagaaatccaagttggt gtcatacatt 1440 tcattttgat gtgaactttt ctttgctttc ctttgttctaagactccatt ttgcaataaa 1500 cgttttgaca gtaaaaaaaa taaaaaagga aaaaaaa 1537<210> SEQ ID NO 13 <211> LENGTH: 972 <212> TYPE: DNA <213> ORGANISM:Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <400> SEQUENCE:13 acgcaaattc ggcacgaggg ttctaaaacc cagtttggtt tacgttgtct ttcacagtag 60tatatttagc tcttctctgg aaagttgtgg gttaatataa ttcttaaaca tgaaaatgta 120attaaacaca ccacgagaga acaatattcc aggagactta atagtgatta ctttcttcaa 180tcaggaaatc gtttcagtgc ctcctttgta ggaatgcttt gttttgtgat gggttttctt 240aaagaagagc acacctccgt ccaatctcct gagacagcca cgtctccgct gacatcccac 300tgtgatgctt tcagatagtc agtgaatgtt tctgataacc ttcatccagt atctgaaaca 360caatgtgaga gattatattg ttttagataa taacatccca tttagttgac taaaatcttc 420caaactctga aagctgcaca ctgctactcc agagagtgca ggtcttagct cttctccttt 480ctgacttcaa gatgaatctt tgggacgatg tttctggtgc ttggtccaca gtgattcact 540tttgaaggag aggccacatg acatgaactg cctggtgtta caacctagct aacatatttg 600atgctactcc tgttgtctgt actgcttatt caagtagtat tctaagttat gttactaaaa 660aacatggtgg gtaaagcaca atcctaccca tcattgtcct ccaaaataat tgtatgacat 720acacggccca gcccattgcc ctccctgcat ctctgtgctg ctttgccatt tccccttcta 780cccagcctcc tcaaggggta ccttggtgga tatttcagta cttaaaacca gactgtaatc 840ataacctccc tctgtgtggc atcaataaat agccaaactc aaaaaaaaaa aaaaaaaaaa 900aaaaaaaaaa aaaaaatatc ggtcgcaagc ttattccctt tagtgagggt taattttagc 960ttgcactgcc ta 972 <210> SEQ ID NO 14 <211> LENGTH: 1544 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature<400> SEQUENCE: 14 tactttgact ttggatcatt tccctgactg ggctaatgtgacacatattg agacttagga 60 agagccacaa gaccacacac acagccctta ccctcctcaggactaccgaa ccttctggca 120 caccttgtac agagttttgg ggttcacacc ccaaaatgacccaacgatgt ccacacacca 180 ccaaaaccca gccaatgggc cacctcttcc tccaagcccagatgcagaga tggacatggg 240 cagctggagg gtaggctcag aaatgaaggg aacccctcagtgggctgctg gacccatctt 300 tcccaagcct tgccattatc tctgtgaggg aggccaggtagccgagggat caggatgcag 360 gctgctgtac ccgctctgcc tcaagcatcc cccacacagggctctggttt tcactcgctt 420 cgtcctagat agtttaaatg ggaatcagat cccctggttgagagctaaga caaccaccta 480 ccagtgccca tgtcccttcc agctcacctt gagcagcctcagatcatctc tgtcactctg 540 gaagggacac cccagccagg gacggaatgc ctggtcttgagcaacctccc actgctggag 600 tgcgagtggg aatcagagcc tcctgaagcc tctgggaactcctcctgtgg ccaccaccaa 660 aggatgagga atctgagttg ccaacttcag gacgacacctggcttgccac ccacagtgca 720 ccacaggcca acctacgccc ttcatcactt ggttctgttttaatcgactg gccccctgtc 780 ccacctctcc agtgagcctc cttcaactcc ttggtcccctgttgtctggg tcaacatttg 840 ccgagacgcc ttggctggca ccctctgggg tcccccttttctcccaggca ggtcatcttt 900 tctgggagat gcttcccctg ccatccccaa atagctaggatcacactcca agtatgggca 960 gtgatggcgc tctgggggcc acagtgggct atctaggtcctccctcacct gaggcccaga 1020 gtggacacag ctgttaattt ccactggcta tgccacttcagagtctttca tgccagcgtt 1080 tgagctcctc tgggtaaaat cttccctttg ttgactggccttcacagcca tggctggtga 1140 caacagagga tcgttgagat tgagcagcgc ttggtgatctctcagcaaac aacccctgcc 1200 cgtgggccaa tctacttgaa gttactcgga caaagaccccaaagtggggc aacaactcca 1260 gagaggctgt gggaatcttc agaagccccc ctgtaagagacagacatgag agacaagcat 1320 cttctttccc ccgcaagtcc attttatttc cttcttgtgctgctctggaa gagaggcagt 1380 agcaaagaga tgagctcctg gatggcattt tccagggcaggagaaagtat gagagcctca 1440 ggaaacccca tcaaggaccg agtatgtgtc tggttccttgggtgggacga ttcctgacca 1500 cactgtccag ctcttgctct cattaaatgc tctgtctcccgcgg 1544 <210> SEQ ID NO 15 <211> LENGTH: 1109 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <220>FEATURE: <221> NAME/KEY: unsure <222> LOCATION: 751-806, 884-885 <223>OTHER INFORMATION: a, t, c, g, or other <400> SEQUENCE: 15 cggacgcgtgggggccagcc tggaggccca gacgtggcgc agcgactcgg aggttcgcct 60 ccagcttgcgcatcatctgc ggccgggtcc cgatgagcct cctgttgcct ccgctggcgc 120 tgctgctgcttctcgcggcg cttgtggccc cagccacagc cgccactgcc taccggccgg 180 actggaaccgtctgagcggc ctaacccgcg cccgggtaga gacctgcggg ggatgacagc 240 tgaaccgcctaaaggaggtg aaggctttcg tcacgcagga cattccattc tatcacaacc 300 tggtgatgaaacacctccct ggggccgacc ctgagctcgt gctgctgggc cgccgctacg 360 aggaactagagcgcatccca ctcagtgaaa tgacccgcga agagatcaat gcgctagtgc 420 aggagctcggcttctaccgc aaggcggcgc ccgacgcgca ggtgcccccc gagtacgtgt 480 gggcgcccgcgaagccccca gaggaaactt cggaccacgc tgacctgtag gtccgggggc 540 gcggcggagctgggacctac ctgcctgagt cctggagaca gaatgaagcg ctcagcatcc 600 cgggaatacttctcttgctg agagccgatg cccgtccccg ggccagcagg gatggggttg 660 gggaggttctcccaacccca ctttcttcct tccccagctc cactaaattc cctcctgcct 720 taaaaaaaacaagaaaaacc aaacaaacaa nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 780 nnnnnnnnnnnnnnnnnnnn nnnnnntctt ctatagtgtc acctaaattc aattcactgg 840 ccgtcgttttacaacgtcgt gactggcgac ggacaaagtt atcnntttaa tcgccttgca 900 gcacatacccctttggccag ctggggtaat aggggaagcg ggccggaccc gatcggcctt 960 cccaaacagttggggaagct tgaaatgcgc gacattgggc cgacggcctt ctatacggga 1020 ggatctctaaacgcggccgg ggtgttggtt gggttaaggc ggagtgtgac cccgcataat 1080 aacttttgcacaggggccct ataggggcc 1109 <210> SEQ ID NO 16 <211> LENGTH: 1740 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:misc_feature <400> SEQUENCE: 16 aagagaagtt accccgatga cttggtttggaaggggttaa ggcaccagtg catcctcttc 60 taaagtgatt tatgatgatg tgtggagtttaaaaacttta ccccacccca aagaacagcc 120 ctctcactcc tcactgagtc cactctgaacgtgctaaaat gggaaggagg cggtgttttg 180 ctgatctgtt aaattcttag tgaagtttccttgatttcca gtggctgctg ttgtttgagt 240 ttggtttgga gcaaaactga ggtagtcctaacatttctgg gactgaatcc aggcaagaga 300 aagaagaaaa agaagaagaa aaagaggaggaaaaaggtag ggagaaataa agggaggaga 360 gaagcacagt gaaagaaaaa aaaagtcccttttgcgacat cacattcctg tgttttccct 420 cagcctggaa aacatattaa tcccagtgcttttacgcccg gaaacaaaga gactaagcca 480 gactatgggg gaaagggaga taagaaggatcctggaactt taaagaggga aagagtgaga 540 ttcagaaatc gccaggactg gactttaagggacgtcctgt gtcagcacaa gggactggca 600 cacacagaca cacgagaccg aggagaaactgcagacaaat ggagatacaa agacttagaa 660 ggacagctcc tttcacctca tcctacttgtccagaaggta aaaagacaca gccagaaaga 720 aaaggcatcg gctcagctct cagatcaggacaggctgtgg atctgtggcg gtactctgaa 780 agctggagct gcagcacacc ccttttgtattgctcaccct cggtaaagag agagagggct 840 gggaggaaaa gtagttcatc taggaaactgtcctgggaac caaacttctg atttcttttg 900 caaccctctg cattccatct ctatgagccaccattggatt acacaatgac atggagaatg 960 ggaccccgtt tcactatgct gttggccatgtggctagtgt gtggatcaga accccacccc 1020 catgccacta ttagaggcag ccacggaggacggaaagtgc ctttggtttc tccggacagc 1080 agtaggccag ctcggtttct gaggcacactgggaggtctc gcggaattga gagatccact 1140 ctggaggaac caaaccttca gcctctccagagaaggagga gtgtgcccgt gttgagacta 1200 gctcgcccaa cagagccgcc agcccgctcggacatcaatg gggccgccgt gagacctgag 1260 caaagaccag cagccagggg ctctccgcgtgagatgatca gagatgaggg gtcctcagct 1320 cggtcaagaa tgttgcgttt cccttacggggtccagctct cccaacatcc ttgccagctt 1380 tgcagggaag aacagagtat gggtcatctcagcccctcat gcctcggaag gctactaccg 1440 cctcatgatg agcctgctga aggacgatgtgtactgtgag ctggcggaga ggcacatcca 1500 acagattgtg ctcttccacc aggcaggtgaggaaggaggc aaggtgagaa ggatcaccag 1560 cgagggccag atcctggagc agcccctgggaccctagcct catccctaag ctgatgagct 1620 tcctgaagct ggagaagggc aagtttggcatggtgctgct gaagaagacg ctgcaggtgg 1680 aggagcgcta tccatatccc gttaggctggaagccatgta cgaggtcatc gaccaaggcc 1740 <210> SEQ ID NO 17 <211> LENGTH:4467 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: misc_feature <220> FEATURE: <221> NAME/KEY: unsure <222>LOCATION: 971, 978, 1295-1522 <223> OTHER INFORMATION: a, t, c, g, orother <400> SEQUENCE: 17 gcgtcgcgct caccctgcgc gtgcccccgc ggcgcctgggcgtcttcctg gactacgagg 60 ccggagagct gtccttcttc aacgtgtccg acggctcccacatcttcacc ttccacgaca 120 ccttctcggg cgcgctctgt gcgtacttca ggcccagggcccacgacggc ggcgaacatc 180 cggatcccct gaccatctgc ccgctgccgg ttagagggacgcgcgtcccc gaagagaacg 240 acagtgacac ctggctacag ccctatgagc ccgcggatcccgccctggac tggtggtgag 300 gcgccctcgt ggccgcggga ctggccccgg gggggccccctggatcccag gccagcgctt 360 tgctctcctg ctccgtctga agggagcagg tgcaccagccaaaatgtcag cgagggggac 420 aaagagaggg acctttgcct acgtagatgt gtatgtgtagtgcgattttc ttcaaggaaa 480 ggagacaagt ccaaagctcg tttgtggatt gtgggactgagcaaaggagt acaaatatat 540 ccacgtcgct cagagctggg gtgctcacgg tgggtggtgggaaagaagcc agcatggaag 600 aaagaaggga gaaaactttg gtgactgcct tagagggatcagttaatttg tatagtttta 660 tattttttgt atatgtttgc tagctctaaa aaggtcgagatgcaataaca cttcgtaagc 720 aacgagttca cctaagtaag gctcagatcc tagttttaaaaaccatttcc cattaaaatg 780 aagttggagg aacagctgct tctggagccg gggcaaaaaatttcaaggtg agcctggagc 840 attgtgtgtg gtgaagtaaa ataaaggctc aaaacgtgacggcaacccgg caaaagggta 900 gggagccagg ccgaagggcc tcactgacca attgtgggacaatttgaaca tcaggatgaa 960 taatgacagg ngaggttnta acacactgaa taaaaacataatccatgagt tcatgctgat 1020 actcaaattt ctttttaaaa aggagaaaca ggaaggtttcttttggaggt gaaatctaat 1080 tattggtgag agtcttggag aacaggctgt ttccagtctcaaagcagtaa ccttatacac 1140 tacttataag tttgaaaggg gaaaggttac ctttacaatggagacatcta ccagatgcat 1200 ccaagtgatt aaatttaaca tcatcaatga tgggaccaaggacattatta gtttgacaac 1260 tggggaaaga agtgttcttc accccctacc cccannnnnnnnnnnnnnnn nnnnnnnnnn 1320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn 1380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn 1440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn 1500 nnnnnnnnnn nnnnnnnnnn nnttcatttt cagagtgagacatttgtact gtggctatgt 1560 aggagaacat tcttgttctt agcaaacata ctgaagtttttagatattaa ttaccacagt 1620 gtctgccact gaatttccag tgactaagtg gaaaaatataaaacatatga atataaagaa 1680 agaaagagac aagtcaaatg tagtaaaatg acaacacttggtgactctag gtgactggtc 1740 gacagatgtt cattgtacta tcaatgtggc tttgctgtgggtttgaaatt ttgcaaacta 1800 agagttgggt ggcggggaga aggatacacc aaaaaactaagtgattatct ttggatggga 1860 aaatgtttgg taattgcatt cttaaaatgt cttctttgtattttttaatg ttcaataatg 1920 tatatgtatc agttctgtaa taaaggggaa aacactttttttaaatactc ataaaaaacc 1980 atccgtagga tcgagaagat caggcagaag ggctttgtccagaaatgtaa ggcctctggt 2040 gtagagggcc aggtggtggc ggaggggaat gacggtggagggggagcagg aaggccaagc 2100 ctgggcagcg agaagaagaa agaggaccca aggagagcacaagtcccacc aaccagagag 2160 agtcgggtga aggtcctgag aaaactggcc gccactgcaccagctttgcc ccaacctccc 2220 tcaaccccca gagccaccac ccttcctcct gccccaggccacaacagtga ctcggtccac 2280 gtcccgggcg gtaacagttg ctgcaagacc tatgaccaccactgcctttc ccaccacggc 2340 agaggccctg gaccccctca ccctcccaca ggccccctacaaccactgag gtgatcactg 2400 ccaggagacc ctcagtttca gagaatcttt accctccatcccggaaggat cagcacaggg 2460 agaggccaca gacaaccagg aggcccagca aggccaccagcttggagagc ttcacaaatg 2520 cccctcccac caccatctca gaacccagca caagggctgctggcccaggc cgtttccggg 2580 acaaccgcat ggacaggcgg gaacatggcc accgagacccaaatgtggtg ccaggtcctc 2640 ccaagccagc aaaggagaaa cctcccaaaa agaaggcccaggacaaaatt cttagtaatg 2700 agtatgagga gaagtatgac ctcagccggc ctactgcctctcagctggag gacgagctgc 2760 aggtggggaa tgttcccctt aaaaaagcaa aggagtctaaaaagcatgaa aagcttgaga 2820 aaccagagaa ggagaagaaa aaaaagatga agaatgagaacgcagacaag ttacttaaga 2880 gtgaaaagca aatgaagaag tctgagaaaa agagcaagcaagagaaagag aagagcaaga 2940 agaaaaaagg aggtaaaaca gaacaggatg gctatcagaaacccaccaac aaacacttca 3000 cgcagagtcc caagaagtca gtggccgacc tgctggggtcctttgaaggc aaacgaagac 3060 tccttctgat cactgctccc aaggctgaga acaatatgtatgtgcaacaa cgtgatgaat 3120 atctggaaag tttctgcaag atggctacca ggaaaatctctgtgatcacc atcttcggcc 3180 ctgtcaacaa cagcaccatg aaaatcgacc actttcagctagataatgag aagcccatgc 3240 gagtggtgga tgatgaagac ttggtagacc aagcgtctcatcagcgagct gaggaaagag 3300 tacggaatga cctacaatga cttcttcatg gtgctaacagatgtggatct gagagtcaag 3360 caatactatg aggtaccaat aacaatgaag tctgtgtttgatctgatcga tactttccag 3420 tcccgaatca aagatatgga gaagcagaag aaggagggcattgtttgcaa agaggacaaa 3480 aagcagtccc tggagaactt cctatccagg ttccggtggaggaggaggtt gctggtgatc 3540 tctgctccta acgatgaaga ctgggcctat tcacagcagctctctgccct cagtggtcag 3600 gcgtgcaatt ttggtctgcg ccacataacc attctgaagcttttaggcgt tggagaggaa 3660 gttgggggag tgttagaact gttcccaatt aatgggagctctgttgttga gcgagaagac 3720 gtaccagccc atttggtgaa agacattcgt aactattttcaagtgagccc ggagtacttc 3780 tccatgcttc tagtcggaaa agacggaaat gtcaaatcctggtatccttc cccaatgtgg 3840 tccatggtga ttgtgtacga tttaattgat tcgatgcaacttcggagaca ggaaatggcg 3900 attcagcagt cactggggat gcgctgccca gaagatgagtatgcaggcta tggttaccat 3960 agttaccacc aaggatacca ggatggttac caggatgactaccgtcatca tgagagttat 4020 caccatggat acccttactg agcagaaata tgtaaccttagactcagcca gtttcctctg 4080 cagctgctaa aactacatgt ggccagctcc attcttccacactgcgtact acatttcctg 4140 cctttttctt tcagtgtttt tctaagacta aataaatagcaaactttcac ctattcatga 4200 gttattattg aaacctcaaa tcataaagac atttaaaagaattgtttttc taactggagg 4260 ggctctagtg ctaaataata gtactgaaaa ttgatattattttccttttc ttatatgaag 4320 gaccttattt ggcatataaa attttataaa atatgtatttaaagcttttt cttatttttt 4380 gtattaattg gtaagtgaaa actctgttaa agatcacaccacaatgtttt caagaaacat 4440 ctgaaaagat aaaacaaaga acaaata 4467 <210> SEQID NO 18 <211> LENGTH: 2965 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<220> FEATURE: <221> NAME/KEY: misc_feature <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: 1473-1777, 2948, 2951, 2961 <223> OTHERINFORMATION: a, t, c, g, or other <400> SEQUENCE: 18 aaacaaagttcaatttagct ggatttctga actatggttt tgaatgttta aagaagaatg 60 atgggtacagttaggaaagt ttttttctta cacccgtgac ttgagggaaa cattgcttgt 120 ctttgagaaattgactgaca tactggaaga gaacaccatt ttatctcagg ttagtgaaga 180 atcagtgcaggtccctgact cttattttcc cagaggccat ggagctgaga ttgagactag 240 ccttgtggttttcacactaa agagtttcct tgttatgggc aacatgcatg acctaatgtc 300 ttgcaaaatccaatagaagt attgcagctt ccttctctgg ctcaagggct gagttaagtg 360 aaaggaaaaacagcacaatg gtgaccactg ataaaggctt tattaggtat atctgaggaa 420 gtgggtcacatgaaatgtaa aaagggaatg aggtttttgt tgttttttgg aagtaaaggc 480 aaacataaatattaccatga tgaattctag tgaaatgacc ccttgacttt gcttttctta 540 atacagatatttactgagag gaactatttt tataacacaa gaaaaattta caattgatta 600 aaagtatccatgtcttggat acatacgtat ctatagagct ggcatgtaat tcttcctcta 660 taaagaataggtataggaaa gactgaataa aaatggaggg atatcccctt ggatttcact 720 tgcattgtgcaataagcaaa gaagggttga taaaagttct tgatcaaaaa gttcaaagaa 780 accagaattttagacagcaa gctaaataaa tattgtaaaa ttgcactata ttaggttaag 840 tattatttaggtattataat atgctttgta aattttatat tccaaatatt gctcaatatt 900 tttcatctattaaattaatt tctagtataa ataagtagct tctatatctg tcttagtcta 960 ttataattgtaaggagtaaa attaaatgaa tagtctgcag gtataaattt gaacaatgca 1020 tagatgatcgaaaattacgg aaaatcatag ggcagagagg tgtgaagatt catcattatg 1080 tgaaatttggatctttctca aatccttgct gaaatttagg atggttctca ctgtttttct 1140 gtgctgatagtaccctttcc aaggtgacct tcagggggat taaccttcct agctcaagca 1200 aggagctaaaaggagcctta tgcatgatct tcccacatat caaaataact aaaaggcact 1260 gagtttggcatttttctgcc tgctctgcta agaccttttt ttttttttac tttcattata 1320 acatattatacatgacatta tacaaaaatg attaaaatat attaaaacaa catcaacaat 1380 ccaggatatttttctataaa actttttaaa aataattgta tctatatatt caattttaca 1440 tcctttttcaaaggctttgt ttttctaaag gcnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1500 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1560 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1620 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1680 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1740 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnatc tgggccttac gtaatatatt 1800 ttcttaatggctgcataata tcacatcaaa taggcatttt tcaaacctct ttccttatta 1860 aacatgtagactatatccat tttttactaa aataaataac atttcagata atatctttgc 1920 actgataatgttgccaagcc atttctaaag tgaccttatc aatttaatta ccattggatg 1980 agggtgttgctttcatcgca ccattgtaga ttgtcttttt tatttcaatt tgcgtttatt 2040 tataactggttgcaaaggta cacagaacac acgctccttc aacttatctt tgataaaccc 2100 aagcaaggatacaaaaagtt ggacgacatt gagtagagtc atggtatacg gtgctgaccc 2160 tacagtatcagtggaaaaga taaggaaaat gtcactactc acctatgtta tgcaaaacag 2220 ttaggtgtgctggggctgga tactgctctt ttacttgagc attggttgat taaagtttag 2280 gtaccatccagggctggtct agagaagtct ttggagttaa ccatgctctt tttgttaaag 2340 aagagagtaatgtgtttatc ctggctcata gtccgtcacc gaaaatagaa aatgccatcc 2400 ataggtaaaatgctgaccta tagaaaaaaa tgaactctac ttttatagcc tagtaaaaat 2460 gctctacctgagtagttaaa agcaattcat gaagcctgaa gctaaagagc actctgatgg 2520 ttttggcataatagctgcat ttccagacct gacctttggc cccaaccaca agtgctccaa 2580 gccccaccagctgaccaaag aaagcccaag ttctccttct gtccttccca caacctccct 2640 gctcccaaaactatgaaatt aatttgacca tattaacaca gctgactcct ccagtttact 2700 taaggtagaaagaatgagtt tacaacagat gaaaataagt gctttgggcg aactgtattc 2760 cttttaacagatccaaacta ttttacattt aaaaaaaaag ttaaactaaa cttctttact 2820 gctgatatgtttcctgtatt ctagaaaaat ttttacactt tcacattatt tttgtacact 2880 ttccccatgttaagggatga tggcttttat aaatgtgtat tcattaaatg ttactttaaa 2940 aataaaanaanaaaaaaaaa naaaa 2965 <210> SEQ ID NO 19 <211> LENGTH: 1734 <212> TYPE:DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:misc_feature <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION:571-899 <223> OTHER INFORMATION: a, t, c, g, or other <400> SEQUENCE: 19cgcctccgga aactgccccc cgggctgctg gccaacttca ccctcctgcg cacccttgac 60cttggggaga accagttgga gaccttgcca cctgacctcc tgaggggtcc gctgcaatta 120gaacggctac atctagaagg caacaaattg caagtactgg gaaaagatct cctcttgccg 180cagccggacc tgcgctacct cttcctgaac ggcaacaagc tggccagggt ggcagccggt 240gccttccagg gcctgcggca gctggacatg ctggacctct ccaataactc actggccagc 300gtgcccgagg ggctctgggc atccctaggg cagccaaact gggacatgcg ggatggcttc 360gacatctccg gcaacccctg gatctgtgac cagaacctga gcgacctcta tcgttggctt 420caggcccaaa aagacaagat gttttcccag aatgacacgc gctgtgctgg gcctgaagcc 480gtgaagggcc agacgctcct gggcagtggc caagtcccag tgagaccagg ggcttgggtt 540gagggtgggg ggtctggtag aacactgcaa nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 600nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 660nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 720nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 780nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 840nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnna 900taatcctgct tttacaggtg aaactcgggg ctgtccatag cggctgggac cccgtttcat 960ccatccatgc ttcctagaac acacgatggg ctttccttac ccatgcccaa ggtgtgccct 1020ccgtctggaa tgccgttccc tgtttcccag atctcttgaa ctctgggttc tcccagcccc 1080ttgtccttcc ttccagctga gccctggcca cactggggct gcctttctct gactctgtct 1140tccccaagtc agggggctct ctgagtgcag ggtctgatgc tgagtcccac ttagcttggg 1200gtcagaacca aggggtttaa taaataaccc ttgaaaactg gatcggatga attggctttc 1260attgtgttcc tagcatcttc tcaaatcaac ttcccaggac tccagggtga aggaggaaaa 1320gaggcatggc ccaggccctg gggtgtggga tatggtctcc ctaggggatg acagttggga 1380tcaatggcct gtgacttctc ctctcccttc ccccatcctg ggacctaact ggaaataaaa 1440ccttgactgt tgcccgggtg tcattttacc agtggatttc tgccagggct tgtgtcctag 1500gagaaggttt aagttaaacc agattgccca ggtctccaaa cgatttgtca tgctgacctg 1560agatcatcga agggggcacc tgcccccggg caaggttgca ggggcaggat ggggctgaag 1620ggatgagcag ggtcccgggc ccacctgctg atacagcatt ggccatgtgg gggctgcaat 1680cggatttgga agaccctggg gcttgggggc atgtccattt tcccagtccc taaa 1734 <210>SEQ ID NO 20 <211> LENGTH: 4005 <212> TYPE: DNA <213> ORGANISM: Homosapiens <220> FEATURE: <221> NAME/KEY: misc_feature <400> SEQUENCE: 20ggacaccgtc tgcagtggag tcactggtgc cgtaaatgtg gccaaggggg ccgtccagac 60gggctgtaga cacggccaag accgtgctga ccggcaccaa ggacacagtc actactgggc 120tcatgggggc agtgaatgtc gccaaaggga ccgtccagac cagtgtggac accaccaaga 180ctgtcctaac tggtaccaag gacaccgtct gcagtggggt gaccggtgct gcgaatgtgg 240ccaagggggc cgtccagggg ggcctggaca ctacaaagtc tgtcctgact ggcactaaag 300acaccgtatc cactgggctc acaggggctg tgaacttggc caaagggact gtccagaccg 360gcgtggacac cagcaagact gtcctgaccg gtaccaagga caccgtctgc agtggagtca 420ctggtgccgt aaatgtggcc aaaggcaccg tccagacagg tgtggacaca gccaagacgg 480tgctgagtgg cgctaaggat gcagtgacta ctggagtcac gggggcagtg aatgtggcca 540aaggaaccgt gcagaccggc gtggacgcct ccaaggctgt gcttatgggt accaaggaca 600ctgtcttcag tggggttacc ggtgccatga gcatggccaa aggggccgtc caggggggcc 660tggacaccac caagacagtg ctgaccggaa ccaaagacgc agtgtccgct gggctcatgg 720ggtcagggaa cgtggcgaca ggggccaccc acactggcct cagcaccttc cagaactggt 780tacctagtac ccccgccacc tcctggggtg gactcaccag ttccaggacc acagacaatg 840gtggggagca gactgccctg agcccccaag aggccccgtt ctctggcatc tccacgcccc 900cggatgtgct cagtgtaggc ccggagcctg cctgggaagc cgcagccact accaagggcc 960ttgcgactga cgtggcgacg ttcacccaag gggccgcccc aggcagggag gacacggggc 1020ttttggccac cacacacggc cccgaagaag ccccacgctt ggcaatgctg cagaatgagt 1080tggaggggct gggggacatc ttccacccca tgaatgcgga ggagcaagct cagctggctg 1140cctcccagcc cgggccaaag gtgctgtcgg cggaacaggg gagctacttc gttcgtttag 1200gtgacctggg tcccagcttc cgccagcggg catttgaaca cgcggtgagc cacctgcagc 1260acggccagtt ccaagccagg gacactctgg cccagctcca ggactgcttc aggctgattg 1320aaaaggccca gcaggctcca gaagggcagc cacgtctgga ccagggctca ggtgccagtg 1380cggaggacgc tgctgtccag gaggagcggg atgccggggt tctgtccagg gtctgcggcc 1440ttctccggca gctgcacacg gcctacagtg gcctggtctc cagcctccag ggcctgcccg 1500ccgagctcca gcagccagtg gggcgggcgc ggcacagcct ctgtgagctc tatggcatcg 1560tggcctcagc tggctctgta gaggagctgc ccgcagagcg gctggtgcag agccgcgagg 1620gtgtgcacca ggcttggcag gggttagagc agctgctgga gggcctacag cacaatcccc 1680cgctcagctg gctggtaggg cccttcgcct tgcccgctgg cgggcagtag ctgtaggagc 1740ctgcaggccc ggcgcggggt cgccctgctc tgtccaggga ggagctgcct cagaactttc 1800tccccgcccc caaacctgga tcggttccct aaagccctag acctttgggg ctgcagctgg 1860ctgagcgccg aggggctgcg gaggcagtga ccttcttaac tgagccaccc cacgccctgc 1920tccgggcctg cctgcatctc ccacctcctc cccagcgctg cctgcccctc tcggagcctg 1980gggtcactca gaccaccagc caagagcctt cccttgaagt ccccaagcaa gcactgcaat 2040taggaaagag aaaaagcagc gtgcccagcc tggaagggca tctgtttgcc ccgctagcaa 2100cccttttata tctagcaggg ctcttccagt cctgcagcac gggcccccag ctatcagcgg 2160tgcaggcagt gctgtggcat cccaggctcc gggcagctcc gttctcatgc tgaaagtggg 2220tctccggcct tagcacacac accttgaggg tcttaagaac cacattccct catagtagaa 2280agtactagaa aaagcgacac tgccatcatc atcccaaggc aggctgctac tgcctttgct 2340gacccccggg gtggcctcac ggtggggaca aagctgccag gagccacagc agccacagct 2400ggggctttgc accagcctgg cttgagactg agcagtttgc agggggtggg gggtgcaaaa 2460aacaagcaaa caggctgctg ctgcctccag ctgcccacca caggcctgcc ccaggcacct 2520ggggctctga ggcccctggg gaggctgggc ccagcagctg cccctggaga acacagacaa 2580aggacttccc cgcagggaac tgtgccctat ggagggatca gacagggctg ggaacagcca 2640cagaggctgc gtgcctatgg cacagccctt cctccgccgc acactccccc tgggtcctca 2700ggcccaccca agcgccgggc tgcagaggaa gcggggctgg ggaggctgca ggcatcagag 2760acactggtgg tggcggaccc ggccgccggg ccccgtgctc tcaggctagc ccaggtcgtg 2820gaggctggca ggctcaggtc gggtgtgaga cgtgccgtgg ctgcgctcag tccagcgggg 2880aggagccgtt cagcccggcc tccccaggaa gccatatccc cactcacccg gtaagagaac 2940cttgtcgtcc cctttccatg ctctcctagg acacgagccc aggaacccca gacccagggg 3000gaggaagggt ggaggggccc caggggtcac catgtgcacc aggggccgtg aggggccggg 3060gcattcagct cagctctgaa ccggggaagc tggcacggca aggactgcct caggtgacgg 3120gccgtgagag gggacgggtc aggagccttc ccaagccttc tcctcagccc gacacccatg 3180gccatcggag gctaggatgc cagacacagc catttgcaga aatcaggcac agtgactgca 3240gctcacgtcc agccaaccaa gcatggggcc gcagctcagg aagtcccttc ccgccacacc 3300acagcctaat tcttactggg acggaggcaa ctcggctacg ctgggcagga cgacaaacac 3360gagacgccac tgtggaatga gcaacttcgg agcacggggt gacttgcttg ggaccgtgcc 3420cacgtgacag ccccttatgc agaggaggaa agagaagccc cgagtgggag gggaacctgt 3480ccaaagtcac acggtgtgtg ggtgacacag ctggggtgag tcgaggctgg cccctgaggc 3540ccatgctccc tgaacgctgg agaccactgt cggctagcag cggctctcag ggaaggcctg 3600gtctccaccc tcccagccta gcctcgcgga ccctcgtcct ccccacatcg gacctgctca 3660cctgcctgga ccctgggctg ccagatgcag gaagcatcaa accccccagc ctcgtgggtg 3720cggggcaggg cgcaggcagc acagcttaga tgccctggtt tgtccctctt gtctcctggg 3780aagagcttgc tcccgcccag ctctcctgcc actggccttt cagggttggg ctgggcccag 3840agtgcctttt agtcgcttct cacggtggcc tgatggctca acccagtccc aaacgggccc 3900agtgacactg ccgactgcac cccagctcag gcccccactg caccagcaat gctagaaaac 3960caagccaata aaagtgattt cttttttcat taaaaaaaaa aaaaa 4005 <210> SEQ ID NO21 <211> LENGTH: 846 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: misc_feature <400> SEQUENCE: 21 caaaaatgagcggggtgtgg tggcccatgc ctgtagtccc agctgctcgg gagactgaat 60 ctcttgagcctgggaagcag aggttgcagt gaactgagat cgcgtcactg cactccagcc 120 tggtgacagagcgagattcc atctcaaaaa aaaaaaacag tatgcacgta caaatttctt 180 aacctgttatcaatgtctga gctacataat tatctttcta gttggagttt gttttaggtg 240 tgtaccaactgacatttcag tttttctgtt tgaagtccaa tgtattagtg actctgtggc 300 tgctctcttcacctgcccct tgtggcctgt ctacaattct aaatggattt tgaactcaat 360 gtcgtcgcttctggtttcct gcatatacca atagcattac ctatgacttt ttttttcctg 420 agctattttcactgagctga gctaatgaac taaaactgag ttatgtttaa tatttgtatc 480 aaatacataaaaggaatact gctttttcct tttgtggctc aaaggtagct gcattttaaa 540 atatttgtgaaaataaaaac ttttgttatt agaaaaaaaa aaaaaaaaaa aaaaaaaaaa 600 aaaaaaaaaaagaaagacca aaaaggaaga gaagggaaaa agaagaagag aaacggaggg 660 acaacgggaaacacagagag cgagccggtg acgaaaagcg ggaaggccaa cgcaggagaa 720 gaaagagaggggggcggcgt cgctcattgt gggagtgtcc tcagagttat gcgagtgggg 780 gatgatgggcaggagtgcta tgcgcccctt tgtatgaggg ggtgcctcaa ttgttgatgg 840 gccggg 846<210> SEQ ID NO 22 <211> LENGTH: 1740 <212> TYPE: DNA <213> ORGANISM:Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <220> FEATURE:<221> NAME/KEY: unsure <222> LOCATION: 216-390, 846-889 <223> OTHERINFORMATION: a, t, c, g, or other <400> SEQUENCE: 22 tacaaagttttaagaaagcc agcatctcag aaaggccttt caaacaagga cacttaatta 60 gccatcttatgtataagaaa agaaatataa agaacatgaa aatttaaaaa cagatttggc 120 agttttataacagtctagga ggtggtgtta ttttttccta ttaagaatta gagggcaggt 180 taggaataaataaaatacag tttgaaaata atgagnnnnn nnnnnnnnnn nnnnnnnnnn 240 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 300 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 360 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn gcacttcccc tactgattgc tgccttctct 420 gtggctacaagggacccaca gaattacagg gaagttacag ggaagcaggt ttcatctcaa 480 tattgggagagatttcaaac aatcacacct gcctgagaag gagtgggctg tcactaggaa 540 tttttattcccagtccgtca ggaattttgt agaagggctt catgtgctgg taccaatagg 600 acaggaagattttaatcagc tttactatct atgttttttt atggaaactg tgtgtatgta 660 tacatacattttccaaaaag aaaaattaaa tgattataga gattatgttt ttcagactac 720 tcacgtatctgcttttctta ctccccacct ctgctgataa ttcctagttc attggttttt 780 cccccacactggaattacct ggggagctta aaaaaccctg atgcctgggt cccaccctca 840 gagatnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnt gatatgaatg 900 cagcctgggcactgggaaat ttaaaaactc cccagataat tactgtgaca gccaaggttg 960 agacctcctagtctagagct ttgctataca cagggtggtc cacaagccag gagcatcagc 1020 atcacttggcagcgcttaga gattcagacc ccagacccac tgaatctgac cctgcatttt 1080 cactagaccccaggtgatca gctgcactag actcaacctc taaaccaaga cctcccaccc 1140 tcacagtctatgatccttta gtgaccctca gctgagtcct gtgctgaact gtgtttgttc 1200 tccttgagcacatgcccgct gaccagggac agactggatg agcaagcaac ctgctggcta 1260 tggagaagagccaggctggg taaatgtttg ctgtgactaa gccaggatca aagaactgcc 1320 tgttgcttgcactggctggc actgagcttg ccactctgtg aactgtgctt ccttcccctg 1380 catggacctgtgcctcagtc actattatcc gcaggccttc tccaagggca gccctctcct 1440 tgtttatccctcttaagcct gcgtgcagga aggcacatta accctgtggc cccctgcagg 1500 caggagggtgttgggtgccc ttacctacct tgcccttttt cttgtaccgt aggctgtgcc 1560 gtttatgagtaagtgatgtg tgtctgtgtg tgtgtctaga agtgctgcac tcaccttgtg 1620 ttattggaggttgtgtaacc ccctagcttt gagcctggtc tcagatgttc cttttcccgt 1680 tctctgtccagccgttaacg cccccagtct gtaataaaag cctatcagcc gtgcacttta 1740 <210> SEQ IDNO 23 <211> LENGTH: 499 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<220> FEATURE: <221> NAME/KEY: misc_feature <400> SEQUENCE: 23ctgggatagc aataacctgt gaaaatgctc ccccggctaa tttgtatcaa tgattatgaa 60caacatgcta aatcagtact tccaaagtct aaatatgact attacaggtc tggggcaaat 120gatgaagaaa ctttggctga taatattgca gcattttcca gatggaagct gtatccaagg 180atgctccgga atgttgctga aacagatctg tcgacttctg ttttaggaca gagggtcagc 240atgccaatat gtgtgggggc tacggccatg cagcgcatgg ctcatgtgga cggcgagctt 300gccactgtga gagcctgtca gtccctggga acgggcatga tgttgagttc ctgggccacc 360tcctcaattg aagaagtggc ggaagctggt cctgaggcac ttcgttggct gctactgtat 420atctacaagg accgagaagt caccaagaag ctagtgcggc aggcagagaa gatgggctac 480aaggccatat ttgtgacag 499 <210> SEQ ID NO 24 <211> LENGTH: 1774 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:misc_feature <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: 1679,1681, 1684-1686, 1691-1693, 1705-1708, 1710, 1714, 1716, 1731, 1740,1744 <223> OTHER INFORMATION: a, t, c, g, or other <400> SEQUENCE: 24ctttcctaga caaggctgaa aggggccaac attatttctg aagacttcat tattggaatt 60ctatgggagt gatctcactg agctattttg gaatagaaat gtggctagtt gcctgacctc 120cctcaatggt ttcacgtggc tttcaaaggg aaggaagggc agtgctgact tttggtaaaa 180tgggcgaaag ggtccatgcc agcaacacaa tcactcaaag tccagatgag ggatcagtaa 240atacaacgtg cctgaaaggt ggcccttgag cacattcctc cggtagacat taacttatta 300aattgattct gattacaaat ataaactttg cccccatctc acccagtaac aatgcaagag 360ttgatgtcag tctataaaag gaagtaggaa ctgtccctgg ctttcaggct ccaacatcct 420ccccctgtca agatgtggca cctcaaactt tgtgcagtcc tcatgatctt cctgttgctg 480ttgggccagg taaggaggga aggatactta tgtgtgtgtg tggagtgtgg agatgatagt 540ggtggtggaa cttgaaagct agattcagtc ctgaggaatg gttcctctgt tctgagtcta 600cagcatctgc ggaatggaat gatcactctt ccaaggtgtg cagcagggtg tcaacacttt 660catatctgaa tgtctttgcc cttacagata gatggctccc caataccaga agtgagttcg 720gcaaagagaa ggccacggag aatgacccca ttttggagag gggtttccct caggcctatt 780ggagcctcct gccgggatga ttctgagtgt atcacaaggc tatgcagaaa aagacgctgt 840tccttaagtg tggcccagga atgatgtaca taccagggaa agaaaggaca gcagtcacct 900ccgacaatgc tccgttctat ggaatattga ttaactgcat tttggctgga gacacccaag 960tgaagcaatc ttgtattttt aatatttaaa ggcagatgta cgctttaaat tggtctccat 1020ttcttcttag aatgttgata tatggataag cataactaaa cttgtcaatt tagagtttat 1080ttttctatgg atactattaa atgtctcaaa ttgaaatttt agcagtctgg aattcaagct 1140tttgagggaa agaaggattc actttgtata ctaaagaaaa aaacagcatt gcccaataat 1200gtgttaactt ctcaatctgg aaagtgtagt gagagctaca taatcaatag ctacgtaatc 1260aacttcagca agttcctaag ctgtggccct ggatcccttc actccatact cttcagggag 1320gtgtcaaagg tggtcaagct tgggaggctg aggcaggaga atggcgtgaa ccgggagacg 1380gacttgcagt gagccgagat ccgccactga ctccagcctg gggaaagagc gagactccgt 1440tcacaaaaaa aaagaatcaa aaaaaaaaag gggagccccc ccttggtatc ggaagaccca 1500gtcctgtaat tcacacaggt tgagttcaag gcattaagcc ctgtaagggc cacttcggcc 1560cctcagagtt gctgttctga tccaacggaa gccgcttaca aatttccctt cggaatttgc 1620ctccggcatt ccctaggggc ggtatttgga agcaaagtcc ttttaacagc cagtgtatnc 1680naannncggg nnngcccttg cggcnnnngn ccananattg ctccttcttc ncctcttctn 1740tttnttcccc cccgtgtcga cagggggtgt ggtc 1774 <210> SEQ ID NO 25 <211>LENGTH: 4158 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: misc_feature <220> FEATURE: <221> NAME/KEY: unsure <222>LOCATION: 3667-3722 <223> OTHER INFORMATION: a, t, c, g, or other <400>SEQUENCE: 25 ctcccacaac aatttcattg ttgttagcat atctatttct ccatacattgtaaaactgta 60 atccttaggt atttctaaaa cataaagagg agaattaagt cagctgcagaacaatggggc 120 tgattcttct gctttttctc tggaaaatct ttcattgctt ttggtggaaatttacctaga 180 ggttacaacc acaggatgta gcttggtctc ttatttgcct ttttgggaaaccaattaaga 240 ttaatacagg ataaaggaaa aaagcaatct attcattata taacacagttgtttgtatta 300 cttgttccct gcaaaggaaa tctgttgaat gcttgcattt tgaattcttttctaatagaa 360 caaccaaaaa aggcttctta tggtgcagca ggaaaaaaga tcatttttatagctttgcat 420 tcttaacata gcatttaaag agcggcatga attagaggaa agacatggaacacacaggta 480 gtcggtttga gatcatcggc ttaaaagtat cctaggatgg taatgacccagaagtatttc 540 cagttgtcta gtggtgtggt atgcaggaat gagaagtgtt ttctttccatttcctgttgg 600 acaggtggca atcttagcag agccactatt tggagttgat aactaaagatgcaaataacg 660 tgactatgcc ttctggtcat cctacgacta tttggagttc tccaaaaccttgtaagaggc 720 atgtcaggca tgcagtaaaa gcatctacaa cttcagctgg gcactggcagcataggtctc 780 atcttggacc atacagtccc actttataga agagagtgga agttctccaaaacaatatcc 840 acaacaaagt ctgacctcac tctgagggag atgggaagtg ggaggaagaaggactaacca 900 gctccctgga gtaagaggaa tttgctttcc ctgtctgccc accaggggctatatgtgcca 960 cctttcaggt tggggccaag gaagtgatgt cagtgtgaca gaagggagagttagacctcc 1020 agacgtcagc ctccctccca tggggtacat tttcaatctg agtgttgttgccttagctgt 1080 gttggtatta gcttgattgg ttggtccgct ggttatgagg tgtagggaggcagtttttgt 1140 ttagttttta ggactttgcc tcttcctttg tccttagcat aatttctaggcagagcatcc 1200 acgaagtcgg ttttcattgc cagctcaaga gcgacaatca tttacgagttcctatgttat 1260 gttaggtgcc ttatgtatat tatcccaaat ccactgcatg gtttaaatacaggcactgga 1320 atataaatga aaaaggtcat tacagtcact gactttctgc aggaccttaaacatttctct 1380 ttccacaagt ttccccttaa tcatgtgtca aacctctctt cctgacgggaatgttgtgct 1440 ataatgaatc tgcataacgc ttgggattct aggaggaagg aaggttccatggacatgtaa 1500 gtacagcata ttcccctcag tcttctagga gggcagagtg aatcccagaactggtaagat 1560 tgggaatctg agcattgcca ctttaatctt agaatattta tcattttgacacatcctgtt 1620 ttttagagag gaaaacaaac acagtttctg cattggtagt gtaaagcataccttgttagg 1680 aacgtgtttt gtaagacaca tttgggttgt cattctagag catgtcaaactttgtacttc 1740 aaaatatatt tagtatgatt gttagtggta acatatatca aggctttgaattaactgttt 1800 tatttaattt tcacaagaag cacttatttt agccatagga aaaccaatctgagctacaaa 1860 tagttcttta aaataagccc aggttattta gctattctag aaagtgccgacttctttcaa 1920 gaagcaggca ttgtaggaca gctgagaatt atcacatagc ctaaattctagcctggcagc 1980 aagagtcaca tctgagatgt ccaaaaaaaa aaaaaaaaaa cacctgatctacattgaaag 2040 ggggtagact aacgtatgtg agaccatttt cctatttgca gttacaaggttaaagaactt 2100 tgaaggtcat tcggctgcta agaggcatgt cgaacactct gtgtggctctttcacagtaa 2160 accctcctaa gagcagaaga cacatggctg ttagtgtctg cgtttagatttaatttctca 2220 aataaaggcc cttggctgcg tatcatttca tccagttata aactagggctcctgcaagca 2280 cccccattct aagggtgaat tattgaaatc agttgctatt tgatgagtcacaactggccc 2340 agcaggcagg gcatttgaag tcatggtcat caaaaagaaa tgattgttttttgaaaagct 2400 aaatgcttaa aatgcttcta gagggaagtc gtggggcgtg tgctcattctctttaaaatc 2460 agggttgttg agtttgtttt taaacatttt tataagttca tgagaaaaaatatataaatt 2520 ctaagaacca acactgtatt cccagaaaca tgaccctcgc tggtcttgggtccacatatc 2580 attggactct gggggacaca aagatgcctg tgacactttg gtgttgccgagttagtcaac 2640 aattattctg ggaaaaagca gaattgaatt cttctctaga tgtcctaccagggttggcca 2700 agggccacaa agcaggctaa taaattccca caggatccag acaccaggcaaaattgctct 2760 aagaagccag ttactgtcat ccctctatgg ttctagaaaa aatagtacaaaaatgacagg 2820 tcatcctatg agcgtcatgc caatgaaacc ccatcttctg gagaagcccttgaatcagaa 2880 ttatcttttt tcttgatgtc gtcagatgca gccagtttct taatttttttaaaaactgta 2940 tgtttctgtg gtatgtatat ttgtacacct aactacctgg cacttggaaatcacagcact 3000 actcagaggc aattgaataa agagaaattt aattttaaat atcaagtcctgtcaaacatt 3060 tctcaaactt ctgattttat caaaggtttg ccagccaata aagtgcatcccaagtataca 3120 ggggagaaag ctagactcct acagggtcct agagtttaag taatttttttgttattaata 3180 taggtaataa tttttctaat ttttattttt tggttccaaa tgtaaagctccttgtgttta 3240 cctctgttta tgtcattctt gacatgttta tctaaattat gtgtgctctgtgacaggtga 3300 aatgtaaatc tgggatccat agtcaagata tcataaggac ctacttcccagcctaccttt 3360 cttcctctac ctgataatga taatactcaa aataacaaca ttcaaaggaaacacaaagaa 3420 atcctgcttt cacatctcct atttcttggg ctccttaata actactgatggtttgttcat 3480 gaaaaaaaat ttttaaatca aaagattgta cttggccctg agttgaaaaaatttcaaaaa 3540 tcaaaagttt gtacttggcc ctgagttgaa aaaaaaaatt cacattctaagaataaacag 3600 aaaaatgttc ttcttggaag taaataacaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa 3660 aaaaaannnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn 3720 nntcttctat agtgtcacct aaattcaatt cactggccgt cgttttacaacgtcgtgact 3780 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccctttcgcaagct 3840 ggcgtaatac gcgaagaggc ccgaaccgtt ggcccttccc aacagttgcgcagcctgaat 3900 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggtggttagtaga 3960 ggtgtgccgt aaaaaataga ataatttttt ttcaagagat gagcagaattgagtaggaat 4020 gattacgggg aggaaaagat ctagaagata gacaatagag aggagagaaaaagagggacg 4080 aggaggctga gaggaaaaga gtagaagcgt gatatgaata tatacagaaacagaaaaagg 4140 agagagggta agacataa 4158 <210> SEQ ID NO 26 <211>LENGTH: 366 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: misc_feature <400> SEQUENCE: 26 Met Ala Leu Arg Phe LeuLeu Gly Phe Leu Leu Ala Gly Val Asp 1 5 10 15 Leu Gly Val Tyr Leu MetArg Leu Glu Leu Cys Asp Pro Thr Gln 20 25 30 Arg Leu Arg Val Ala Leu AlaGly Glu Leu Val Gly Val Gly Gly 35 40 45 His Phe Leu Phe Leu Gly Leu AlaLeu Val Ser Lys Asp Trp Arg 50 55 60 Phe Leu Gln Arg Met Ile Thr Ala ProCys Ile Leu Phe Leu Phe 65 70 75 Tyr Gly Trp Pro Gly Leu Phe Leu Glu SerAla Arg Trp Leu Ile 80 85 90 Val Lys Arg Gln Ile Glu Glu Ala Gln Ser ValLeu Arg Ile Leu 95 100 105 Ala Glu Arg Asn Arg Pro His Gly Gln Met LeuGly Glu Glu Ala 110 115 120 Gln Glu Ala Leu Gln Asp Leu Glu Asn Thr CysPro Leu Pro Ala 125 130 135 Thr Ser Ser Phe Ser Phe Ala Ser Leu Leu AsnTyr Arg Asn Ile 140 145 150 Trp Lys Asn Leu Leu Ile Leu Gly Phe Thr AsnPhe Ile Ala His 155 160 165 Ala Ile Arg His Cys Tyr Gln Pro Val Gly GlyGly Gly Ser Pro 170 175 180 Ser Asp Phe Tyr Leu Cys Ser Leu Leu Ala SerGly Thr Ala Ala 185 190 195 Leu Ala Cys Val Phe Leu Gly Val Thr Val AspArg Phe Gly Arg 200 205 210 Arg Gly Ile Leu Leu Leu Ser Met Thr Leu ThrGly Ile Ala Ser 215 220 225 Leu Val Leu Leu Gly Leu Trp Asp Tyr Leu AsnGlu Ala Ala Ile 230 235 240 Thr Thr Phe Ser Val Leu Gly Leu Phe Ser SerGln Ala Ala Ala 245 250 255 Ile Leu Ser Thr Leu Leu Ala Ala Glu Val IlePro Thr Thr Val 260 265 270 Arg Gly Arg Gly Leu Gly Leu Ile Met Ala LeuGly Ala Leu Gly 275 280 285 Gly Leu Ser Gly Pro Ala Gln Arg Leu His MetGly His Gly Ala 290 295 300 Phe Leu Gln His Val Val Leu Ala Ala Cys AlaLeu Leu Cys Ile 305 310 315 Leu Ser Ile Met Leu Leu Pro Glu Thr Lys ArgLys Leu Leu Pro 320 325 330 Glu Val Leu Arg Asp Gly Glu Leu Cys Arg ArgPro Ser Leu Leu 335 340 345 Arg Gln Pro Pro Pro Thr Arg Cys Asp His ValPro Leu Leu Ala 350 355 360 Thr Pro Asn Pro Ala Leu 365

What is claimed is: 1 A combination comprising a plurality of cDNAswherein the cDNAs are SEQ ID NOs:1-25 and the complements of SEQ IDNOs:1-25.
 2. An isolated cDNA comprising a polynucleotide having thenucleic acid sequence of SEQ ID No:8.
 3. A method for detectingdifferential expression of one or more cDNAs in a sample containingnucleic acids, the method comprising: a) hybridizing the combination ofclaim 1 with nucleic acids of the sample, thereby forming one or morehybridization complexes; b) detecting the hybridization complexes; andc) comparing the hybridization complexes with those of a standard,wherein differences between the standard and sample hybridizationcomplexes indicate differential expression of cDNAs in the sample. 4.The method of claim 3, wherein the nucleic acids of the sample areamplified prior to hybridization.
 5. The method of claim 3, wherein thesample is from a subject with a disorder associated withatherosclerosis.
 6. The method of claim 3, wherein the sample is fromaorta, arteries, arterioles, endothelial cells, plaque, or blood.
 7. Themethod of claim 3 wherein the combination is immobilized on a substrate.8. The method of claim 3 wherein differential expression is diagnosticof angina pectoris, coronary artery disease, myocardial infarction,hypertension, transient cerebral ischemia, mesenteric ischemia,peripheral vascular disease, renal artery stenosis, or stroke.
 9. Themethod of claim 6 wherein the substrate is a microarray.
 10. A method ofusing cDNAs to screen a sample to identify a ligand which specificallybinds a cDNA, the method comprising: a) combining the combination ofclaim 1 with the sample under conditions to allow specific binding; andb) detecting specific binding between each cDNA and at least ligand,thereby identifying a ligand that specifically binds to each cDNA. 11.The method of claim 10 wherein the ligands are DNA molecules, proteins,RNA molecules, or transcription factors.
 12. A vector containing thecDNA of claim
 2. 13. A host cell containing the vector of claim
 12. 14.A method for producing a protein, the method comprising the steps of: a)culturing the host cell of claim 13 under conditions for expression ofprotein; and b) recovering the protein from the host cell culture.
 15. Aprotein comprising a polypeptide having the amino acid of SEQ ID NO:26.16. A method for using a protein to screen a plurality of molecules orcompounds to identify at least one ligand which specifically binds theprotein, the method comprising: a) combining the protein encoded by acDNA of claim 1 with the plurality of molecules or compounds underconditions to allow specific binding; and b) detecting specific bindingbetween the protein and a molecule or compound, thereby identifying aligand which specifically binds the protein.
 17. The method of claim 16wherein the plurality of molecules or compounds is selected fromagonists, antagonists, antibodies, DNA molecules, small molecule drugs,immunoglobulins, inhibitors, mimetics, peptide nucleic acids, peptides,pharmaceutical agents, proteins, RNA molecules, and ribozymes.
 18. Amethod of using a protein to produce an antibody, the method comprising:a) immunizing an animal with the protein encoded by a cDNA of claim 1under conditions to elicit an antibody response; b) isolating animalantibodies; and c) screening the isolated antibodies with the protein,thereby identifying an antibody which specifically binds the protein.19. A antibody produced by the method of claim
 18. 20. A method forusing an antibody to detect gene expression in a sample, the methodcomprising: a) combining the antibody of claim 19 with a sample underconditions which allow the formation of antibody:protein complexes; andb) detecting complex formation, wherein complex formation indicatesexpression of the protein in the sample.